Dorsal Unpaired Median Cells Research Articles

Motoneurons, DUM cells, and sensory neurons in an insect thoracic ganglion: A tracing study in the stick insect Carausius morosus

Anatomical features of leg motoneurons, dorsal unpaired median (DUM) cells, and sensory neurons in stick insect mesothoracic ganglia were examined using fluorescent dye backfills of lateral nerves. Structures were analyzed in whole-mounts of ganglia and transverse sections. Numbers of motoneurons and details of their structure by far exceed previously published data. The general neuroanatomical layout of motoneurons matches the general orthopteran pattern. Cell bodies of excitatory motoneurons form clusters in the lateral cortex, dendrites branch mainly in the dorsal neuropil. We identified nine DUM cells, six of which have axons in nerve nl5. Most sensory fibers terminate in the ventral association center (VAC). Twenty-three small cell bodies located close to the soma of the fast extensor tibiae motoneuron likely belong to strand receptors. Labeled structures are compared with previously published data from stick insects and other orthopterous insects.

Activity of neuromodulatory neurones during stepping of a single insect leg

Octopamine plays a major role in insect motor control and is released from dorsal unpaired median (DUM) neurones, a group of cells located on the dorsal midline of each ganglion. We were interested whether and how these neurones are activated during walking and chose the semi-intact walking preparation of stick insects that offers to investigate single leg-stepping movements. DUM neurones were characterized in the thoracic nerve cord by backfilling lateral nerves. These backfills revealed a population of 6–8 efferent DUM cells per thoracic segment. Mesothoracic DUM cells were subsequently recorded during middle leg stepping and characterized by intracellular staining. Seven out of eight identified individual different types of DUM neurones were efferent. Seven types except the DUMna nl2 were tonically depolarized during middle leg stepping and additional phasic depolarizations in membrane potential linked to the stance phase of the middle leg were observed. These DUM neurones were all multimodal and received depolarizing synaptic drive when the abdomen, antennae or different parts of the leg were mechanically stimulated. We never observed hyperpolarising synaptic inputs to DUM neurones. Only one type of DUM neurone, DUMna, exhibited spontaneous rhythmic activity and was unaffected by different stimuli or walking movements.

Ca2+ currents in central insect neurons: electrophysiological and pharmacological properties.

Ca2+ currents in dorsal unpaired median (DUM) neurons isolated from the fifth abdominal ganglion of the cockroach Periplaneta americana were investigated with the whole cell patch-clamp technique. On the basis of kinetic and pharmacological properties, two different Ca2+ currents were separated in these cells: mid/low-voltage-activated (M-LVA) currents and high-voltage-activated (HVA) currents. M-LVA currents had an activation threshold of -50 mV and reached maximal peak values at -10 mV. They were sensitive to depolarized holding potentials and decayed very rapidly. The decay was largely Ca2+ dependent. M-LVA currents were effectively blocked by Cd2+ median inhibiting concentration (IC50 = 9 microM), but they also had a remarkable sensitivity to Ni2+ (IC50 = 19 microM). M-LVA currents were insensitive to vertebrate LVA channel blockers like flunarizine and amiloride. The currents were, however, potently blocked by omega-conotoxin MVIIC (1 microM) and omega-agatoxin IVA (50 nM). The blocking effects of omega-toxins developed fast (time constant tau = 15 s) and were fully reversible after wash. HVA currents activated positive to -30 mV and showed maximal peak currents at + 10 mV. They were resistant to depolarized holding potentials up to -50 mV and decayed in a less pronounced manner than M-LVA currents. HVA currents were potently blocked by Cd2+ (IC50 = 5 microM) but less affected by Ni2+ (IC50 = 40 microM). These currents were reduced by phenylalkylamines like verapamil (10 microM) and benzothiazepines like diltiazem (10 microM), but they were insensitive to dihydropyridines like nifedipine (10 microM) and BAY K 8644 (10 microM). Furthermore, HVA currents were sensitive to omega-conotoxin GVIA (1 microM). The toxin-induced reduction of currents appeared slowly (tau approximately 120 s) and the recovery after wash was incomplete in most cases. The dihydropyridine insensitivity of the phenylalkylamine-sensitive HVA currents is a property the cockroach DUM cells share with other invertebrate neurons. Compared with Ca2+ currents in vertebrates, the DUM neuron current differ considerably from the presently known types. Although there are some similarities concerning kinetics, the pharmacological profile of the cockroach Ca2+ currents especially is very different from profiles already described for vertebrate currents.

Distribution of myomodulin-like immunoreactivity in the adult and developing ventral nervous system of the locust Schistocerca gregaria.

The distribution of myomodulin-like immunoreactivity in the ventral nervous system of an insect, the locust Schistocerca gregaria, both in the adult and during development, is described. The results suggest the presence of a novel modulatory system in insects which uses myomodulin-like neuropeptides. The study also indicates that the myomodulins, which were first identified in mollusks, may represent another interphyletic family of neuropeptides. In the suboesophageal ganglion, immunoreactive cells occur in five groups. The processes from the two anterior ventral midline groups of cells project to the corpora allata via nervi corpora allata II. Thus myomodulin-like neuropeptides may be involved in the control of the release of juvenile hormone from the corpora allata. The thoracic ganglia contain three groups of immunoreactive cells, including a bilaterally symmetrical group of 12-15 posterior lateral cells, which project to the median nerve and its neurohaemal organs, suggesting a possible neurohaemal role for myomodulin-like peptides. Each thoracic neuromere also contains a single, intensely stained, dorsal unpaired median (DUM) cell that may correspond to the so-called H cell. In the abdominal ganglia, the staining shows sexual dimorphism, both in terms of the number of dorsal and ventral midline cells stained and in terms of the distribution of their immunoreactive processes. Myomodulin-like immunoreactivity is one of the earliest neurotransmitter/neurohormone phenotypes detectable during the development of the locust nervous system. It first appears in the single DUM cells in each of the thoracic neuromeres at 50% development, and the complete adult pattern of staining is present at 85-90% of development.

Ca2+ currents in cockroach neurones: properties and modulation by neurohormone D.

In dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana two types of Ca2+ currents were found: a transient one activated by lower depolarizations (LVA) and a sustained one activated by higher voltage jumps (HVA). Both currents were strongly reduced by 100 microM Cd2+; Ni2+ was less effective. The HVA currents were insensitive to dihydropyridines, but sensitive to phenylalkylamines. The LVA component was sensitive to omega-conotoxin GVIA and SK&F96365 whereas the HVA component was insensitive to both blockers. The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), is known to increase the spiking frequence in these DUM cells. We demonstrate that NHD increases the LVA current component and decreases the HVA current component. These effects of NHD can explain the previously found NHD-induced stimulation of Ca2+ dependent K+ currents.

Neurohormone D induces ionic current changes in cockroach central neurones.

The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), increases the frequency of spontaneous activity in dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana. The increase in spike frequency is accompanied by changes in the shape and the amplitude of the single action potentials, e.g. a more pronounced afterhyperpolarization. Effects of NHD on membrane currents were investigated in these DUM cells with whole-cell voltage-clamp measurements. A voltage-independent Ca2+ current flowing at the resting potential (ICa,R) was found. NHD, at nanomolar concentrations, enhanced this ICa,R in a concentration-dependent manner. 0.1 mM Cd2+ markedly reduced ICa,R and in this case ICa,R was hardly potentiated by NHD. In the presence of NHD a fast activating Ca(2+)-dependent K+ current sensitive to charybdotoxin and to low concentrations of tetraethylammonium was augmented. The enhanced afterhyperpolarization of action potentials can be accounted for by the increase in the Ca(2+)-dependent K+ current. The changes of the membrane currents induced by NHD are discussed with respect to further effects on the spike pattern and in relation to the previously described mode of action of AKH-peptides in other preparations.

Neuritic GABAergic synapses in insect neurosecretory cells

The localization of synaptic γ-aminobutyric acid (GABA) receptors on cockroach dorsal unpaired median (DUM) neurons of the last abdominal ganglion was investigated. These neurosecretory cells, mainly octopaminergic, possess a soma located on the dorsal midline of the ganglion, from which emerges a short primary neurite dividing into two symmetrical lateral branches on both lateral edges of the ganglion. GABA pressure ejections onto the soma and onto the neuritic arborization elicited hyperpolarizations. Moreover, electrical stimulation of the anterior connectives evoked a postsynaptic potential, mainly inhibitory. This response and the GABA hyperpolarization of the neuritic field are antagonized by lateral application of picrotoxin while soma GABA hyperpolarization remained unchanged. This suggests that there are two kinds of GABA receptors located (i) onto the soma membrane of the DUM neurons and called extrasynaptic receptors, (ii) on the neuritic arborization, called synaptic receptors and implicated in the connections between neurons coming from the anterior part of the nervous system and the DUM cells. Immunohistological double staining technique reinforced the electrophysiological results by showing the presence of GABA-like immunoreactive processes next to octopamine-like immunoreactive ones.

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 octapamine-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 ganglia, this investigation shows that colocalization of octopamine with an FMRFamide related peptide is restricted to a single DUM cell occuring in each abdominal ganglion 2–7, which most likely correponds to segmental homologues of DUM heart-1.

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.

Suboesophageal DUM neurons innervate the principal neuropiles of the locust brain

The morphology of the dorsal unpaired median (DUM ) neurons of the suboesophageal ganglion (SOG) of the migratory locust, Locusta migratoria , were studied by using intracellular staining. The SOG lacks segmental DUM neurons with peripheral axons. All DUM neurons are either intersegmentally projecting (towards the brain or the thoracic nerve cord) or they are local. In addition to previously described DUM neurons with axons in peripheral nerves of the brain (Bräunig 1990), the SOG contains DUM neurons which, in the brain, innervate principal neuropile areas such as the antennal lobes, the pedunculi and calyces of the mushroom body, and the central complex. The number and location of DUM cell bodies stained with intracellular fills is compared with those obtained with either backfilling cervical or circumoesophageal connectives, or octopamine-immunocytochemistry. Additional experiments show that the locust brain, like the SOG, lacks both segmental DUM neurons with peripheral axons, and axons descending into the ventral nerve cord.

Sulphide Silver Staining for Endogenous Heavy Metals Reveals Subsets of Dorsal Unpaired Median (Dum) Neurones In Insects

ABSTRACT The central nervous systems of many insects contain segmentally arranged groups of unpaired midline neurones. These have been called DUM (dorsal unpaired median) neurones because of the position of their cell bodies. The larger DUM neurones, with bilaterally symmetrical peripheral axons, have been most extensively studied in grasshoppers and locusts. DUM cells in the metathoracic ganglion modulate tension of skeletal muscle, potentiate synaptic transmission at neuromuscular junctions and slow a myogenic rhythm (Evans and O’Shea, 1977, 1978; Whim and Evans, 1989), whereas those in the abdominal ganglia alter tension in visceral muscle (Lange and Orchard, 1984). In fireflies, DUM neurones of the terminal abdominal ganglia control flashing by activating the photocytes of the lantern (Christensen and Carlson, 1981). The effects are mediated by the biogenic amine octopamine (Carlson and Jalenak, 1986; Evans and O’Shea, 1977, 1978; Morton and Evans, 1984; Orchard and Lange, 1985), which is also implicated in other modulatory actions (see Ramirez and Orchard, 1990, for references). In grasshoppers, in addition to the octopamine-containing efferent DUM neurones, there are a substantial number of local and intersegmental DUM interneurones with small cell bodies (Thompson and Siegler, 1989, 1991).

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

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.

Octopaminergic modulation of the forewing stretch receptor in the locust Locusta Migratoria

ABSTRACT Modulatory actions of various biogenic amines and peptides on the locust forewing stretch receptor (SR) were examined. The response of the SR to sinusoidal wing movements was unaffected by physiological concentrations (5×10−8mol l−1) of the peptides AKHI, AKHII, proctolin and FMRFamide. The biogenic amine octopamine, however, enhanced the SR response in a dosedependent manner when injected into the haemolymph of an almost intact animal or perfused over an isolated thorax preparation in which head, abdomen, gut and the entire central nervous system were removed (threshold at 5×10−8mol l−1, maximal effect at 5×10−4mol l−1 DL-octopamine). The SR was as sensitive to D-octopamine, the naturally occurring isomer of octopamine, as it was to DL-octopamine. Serotonin was equal to octopamine in effectiveness, followed in order of potency by synephrine, metanephrine and tyramine. Dopamine was ineffective. Phentolamine, but not DL-propranolol, antagonized the action of octopamine. The threshold of the modulatory effect of octopamine on the SR suggests that the increased haemolymph octopamine level which occurs during flight is sufficient to increase the SR activity. Two observations suggest that dorsal unpaired median (DUM) cells are involved in the octopaminergic modulation of the SR during flight: (1) selective stimulation of these cells modulated the SR response and this effect was blocked by phentolamine; and (2) a number of DUM cells were activated during flight. These results suggest that the SR activity is enhanced by octopamine following the onset of flight. Since the SR is involved in the control of wing beat frequency, the modulation of the SR might influence the generation of the motor pattern in flying locusts.

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.

Monoclonal antibodies to the cockroach nervous system.

In the cockroach nervous system individual motor neurons may be identified with respect to their position in the thoracic ganglia and to the muscles they innervate. When their axons are cut they have the ability to regrow such that when regeneration is completed they have specifically reinnervated their normal target muscles. This suggests the existence of a specific intercellular recognition process between motor neurons and muscles, and that neurons innervating different muscles are biochemically distinct from one another. The goal of this study was to use hybridoma techniques to obtain monoclonal antibodies that bind to some motor neurons and not others. Mice were injected with whole nerve cord and hybridoma supernatants were screened immunohistochemically on sections of ganglion and leg muscle. The monoclonal antibodies were categorized according to four types of specificity: tissue, regional, cell-type, and neuron-subset specificities. Antibodies expressing neuron-subset specificity were obtained very rarely. The probability of their occurrence could be increased by treating the mice with immunosuppressant drugs after initial administration of immunogen or by fixing the immunogen with paraformaldehyde in a manner similar to that of the tissue sections used in the screening process. Two of the neuron-subset specific monoclonal antibodies (MAbs) are of particular interest with respect to the goals of this study. They bind to axon terminals in the muscles of some neurons and not others. They do not bind to neuronal cell bodies in the ganglion, which makes identification of the neurons difficult. However, from the known innervation pattern of the coxal depressor muscles it appears that one of these MAbs selectively binds to axon terminals from either the inhibitory motor neurons or the dorsal unpaired median cells. Other antibodies of interest bind selectively to the synapse-rich neuropile in the ganglia or to peripheral parts of the nervous system like the nerve roots.

Central and peripheral neurosecretory pathways to an insect flight motor nerve.

Ultrastructural examination of the IIN1b nerve to the dorsal longitudinal flight muscle of Manduca sexta L. verified the presence of neurosecretory processes. Subspherical and irregular vesicles were found where the nerve enters the muscle, while spherical vesicles were found in the proximal region only. A dorsal unpaired median (DUM) cell, the median nervous system, and two or more peripheral cells are the sources of these neurosecretory inclusions. Light the electron microscopy CoCl2 backfills of the transverse nerve produced intensification of a peripheral neuron (#1) and processes in nerves IIN1a and IIN1b. Similar backfills of nerve IIN1b produced intensification of a DUM cell, a second peripheral neuron (#2), and processes in the transverse nerve and nerve IIN1a. Neuron #1 contained large spherical electron-dense vesicles while neuron #2 contained smaller subspherical vesicles. These cells were situated upon the link and/or transverse nerves. Based on these results, we suspect central and peripheral neurosecretory processes reach nerve IIN1b as follows: the link nerve projects prothoracic median nervous system and neuron #2 processes, nerve IIN1a projects neuron #1 processes, and nerve IIN1 projects mesothoracic DUM cell processes, although this latter pathway was less clear.

Lectin receptors in the cockroach neuromuscular system. III. Distribution and identification within the thoracic ganglia of the nervous system

A fluorescence microscopic study of the binding of an array of 10 fluorescein isothiocyanate conjugated lectins to frozen sections of the cockroach thoracic ganglia was performed. Although a region of the neuropile receiving direct input from sensory neurons was observed to have distinctive lectin binding properties, no difference was detected between the lectin binding properties of D f and D s, the two identified motor neurons that innervate the coxal depressor muscles in the leg. In addition, the three types of neurons identified in the ganglia, excitatory motor neurons, inhibitory motor neurons and dorsal unpaired median cells all had identical lectin binding properties. Therefore, in order to demonstrate the existence of macromolecules responsible for giving a biochemical identity to the various neurons it will be necessary to perform biochemical analyses of single cells or apply immunological techniques. A biochemical analysis of the ganglionic Con A and WGA receptors detected after fractionation by SDS polyacrylamide gel electrophoresis revealed the presence of some receptors not found in extracts of muscles and which may be specific for ganglia. In addition, a set of ganglionic lectin receptors were detected which were of similar solubility and lectin binding properties to a set of receptors found in muscle extracts and which disappeared from the muscles within one week of their denervation. It is suggested that such lectin receptors are found in all motor neurons and are transported to axon terminals within the muscles.

Synthesis of octopamine by insect dorsal median unpaired neurons.

Dorsal unpaired median (DUM) neurons of locusts and grasshoppers inhibit the instrinsic rhythm of contraction of metathoracic extensor tibia muscle fibers. This physiological action is mimicked by very low concentrations of octopamine and by higher concentrations of dopamine and noradrenaline. We have examined the synthesis of biogenic amines from tritiated tyrosine by these neurons. DUM cell bodies and a peripheral nerve containing the DUM axon which terminates in the extensor tibia both produced octopamine, but neither noradrenaline nor dopamine synthesis was detected. The observations support the suggestion that the dorsal unpaired median neurons are octopaminergic.