Ion currents and spiking properties of identified subtypes of locust octopaminergic dorsal unpaired median neurons.

Efferent dorsal unpaired median (DUM) neurons are key elements of an insect neuromodulatory system. In locusts, subpopulations of DUM neurons mediate octopaminergic modulation at specific targets depending on their activity during different behaviours. This study investigates whether in addition to synaptic inputs, activity in DUM neurons depends on intrinsic membrane properties. Intracellular in situ recordings and whole-cell patch-clamp recordings from freshly isolated somata characterize somatic voltage signals and the underlying ion currents of individual subtypes of DUM neurons identified beforehand by a vital retrograde tracing technique. Na(+), Ca(2+), K(+) currents and a hyperpolarization-activated (I(h)) current are described in detail for their (in-)activation properties and subtype-specific current densities. In addition, a Ca(2+)-dependent K(+) current is demonstrated by its sensitivity to cadmium and charybdotoxin. This complex current composition determines somatic excitability similar in all subtypes of DUM neurons. Both Na(+) and Ca(2+) currents generate overshooting somatic action potentials. Repolarizing K(+) currents, in particular transient, subthreshold-activating A-currents, regulate the firing frequency and cause delayed excitation by shunting depolarizing input. An opposing hyperpolarization-activated (I(h)) current contributes to the resting membrane potential and induces rebound activity after prolonged inhibition phases. A quantitative analysis reveals subtype-specific differences in current densities with more inhibitory I(K) but less depolarizing I(Na) and I(h) - at least in DUM3 neurons promoting a reliable suppression of their activity as observed during behaviour. In contrast, DUM neurons that are easily activated during behaviour (DUM3,4,5 and DUMETi) express less I(K) and a pronounced depolarizing I(h) promoting excitability.

Neuromodulatory octopaminergic neurons and their functions during insect motor behaviour. The Ernst Florey memory lecture.

In this article we describe recent advances in functional studies on the role of octopamine released in the periphery by efferent dorsal or ventral unpaired median neurons. In addition to the previously described modulatory effects on the neuromuscular junction, we describe a metabolic regulatory role for these neurons. Due to their activity glycolytic rates in target tissues, such as muscles, are increased. In flight muscles that use carbohydrate catabolism only at take-off but have to switch to lipid oxidation during prolonged flight, these neurons are only active at rest but are inhibited as soon as flight motor patterns are selected.

Cholinergic transmission via central synapses in the locust nervous system.

In this study we examine the nature of chemical synaptic transmission between identified filiform hair receptors on the prothoracic segment of a locust and the identified postsynaptic projection interneuron (A4I1). The effects of pressure ejected acetylcholine, and various ligands of acetylcholine receptors on the activity of the postsynaptic neuron A4I1, or on wind-elicited responses in A4I1 are reported. It is suggested that the transmitter of the afferent fibers is acetylcholine, and that fast transmission is mediated by nicotinic acetylcholine-receptors. Both nicotine and carbachol act as agonists, whereas d-tubocurarine and alpha-bungarotoxin act as antagonists. The presence of muscarinic acetylcholine receptors was also evident from the modulatory effects of muscarine, oxotremorine and pilocarpine, which were blocked by bath application of atropine. GABA, and its agonists muscimol and cis-4-amino-crotonic-acid lead to inhibition of A4I1 responses. This inhibition was prevented by the additional application of picrotoxin. This suggests involvement of a ligand-gated GABA receptor which, most likely, increases chloride conductance. Metabotropic GABA-receptors do not seem to be involved, since baclofene, diazepam and bicuculline ejections had no effects. Glutamate also inhibits wind elicited A4I1 responses. Although attempts were made to further characterize the receptor involved, tested substances such as kainic acid, glycine, CNQX or GDEE had no effect.

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.

Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling.

We show here, using locust wholemount ganglia as an example, that scaling artifacts in three-dimensional reconstructions from confocal microscopic images due to refractive index mismatch in the light path and tissue shrinking, can account for dramatic errors in measurements of morphometric values. Refractive index mismatch leads to considerable alteration of the axial dimension, and true dimensions must be restored by rescaling the Z-axis of the image stack. The appropriate scaling factor depends on the refractive indices of the media in the light path and the numerical aperture of the objective used and can be determined by numerical simulations, as we show here. In addition, different histochemical procedures were tested in regard to their effect on tissue dimensions. Reconstructions of scans at different stages of these protocols show that shrinking can be avoided prior to clearing when dehydrating ethanol series are carefully applied. Fixation and mismatching buffer osmolarity have no effect. We demonstrate procedures to reduce artifacts during mounting and clearing in methyl salicylate, such that only isometric shrinkage occurs, which can easily be corrected by rescaling the image dimensions. Glycerol-based clearing agents produced severe anisometric and nonlinear shrinkage and we could not find a way to overcome this.

Neuromodulation during motor development and behavior.

Important recent advances have been made in understanding the role of aminergic modulation during the maturation of Xenopus larvae swimming rhythms, including effects on particular ion channel types of component neurons, and the role of peptidergic modulation during development of adult central patterns generators in the stomatogastric ganglion of crustaceans. By recording from octopaminergic neuromodulatory neurons during ongoing motor behavior in the locust, new insights into the role of this peripheral neuromodulatory mechanism have been gained. In particular, it is now clear that the octopaminergic neuromodulatory system is automatically activated in parallel to the motor systems, and that both excitation and inhibition play important functional roles.

Neuroethology, its roots and future.

Scholars in a particular scientific field should be familiar with its historical roots. Such knowledge will put their own research into a historical perspective, and, in addition, will allow them to assess current strengths and weaknesses in their particular area of research. To keep an exciting field like neuroethology alive and close to fast moving scientific frontiers, it is necessary to constantly adapt and broaden its approaches to newly emerging ideas from other fields, and to quickly incorporate new methodologies. The following article tries to expose some of the roots of neuroethology, and, in addition, will present some evidence as to why the authors think this field needs a broader definition than that formulated in the past. Doing so after the 5th International Congress of Neuroethology in San Diego in August 1998 seems to the authors the most appropriate time.

Peripheral distribution of presynaptic sites of abdominal motor and modulatory neurons in Manduca sexta larvae.

Insect muscle fibers are commonly innervated by multiple motor neurons and efferent unpaired median (UM) neurons. The role of UM neurons in the modulation rather than rapid activation of muscle contraction (Evans and O'Shea [1977] Nature 270:257-259) suggests that their terminal varicosities may differ structurally and functionally from the presynaptic terminals of motor neurons. Furthermore, differences in the characteristics of UM neuron terminal varicosities may be correlated with functional differences among their diverse target muscles. Larval abdominal body wall muscles in the hawkmoth, Manduca sexta, consist of large, elongated fibers that are multiterminally innervated by one and occasionally two motor neurons (Levine and Truman [1985] J. Neurosci. 5:2424-2431). The fibers are also innervated by one of two efferent UM neurons that bifurcate to innervate targets on both sides of the abdomen (Pflüger et al. [1993] J. Comp. Neurol. 335:508-522). In this study, the intracellular tracer biocytin was used to identify the targets of the UM neurons and to distinguish their terminal axonal varicosities on the muscle fibers. An antiserum to the synaptic vesicle protein, synaptotagmin, was used to label synaptic vesicles, and the styryl dye FM1-43 was used to demonstrate release and recycling. Most of the abdominal muscles in a given hemisegment were found to be supplied by one of the two UM neurons. Terminal varicosities of the excitatory motor neurons were large (3-7 pm) and were found in rows of rosettes that extended to every aspect of the muscle fiber; these varicosities were designated as type I terminals. The UM neuron terminal varicosities also occupied every aspect of the fiber but were smaller (1-3 microm) and more separated from each other; these were designated as type II terminals. Both type I and type II terminals are synaptotagmin immunoreactive and, as shown by FM1-43 staining, are sites of synaptic vesicle recycling. The excitatory motor neuron terminals (type I) could easily be loaded and unloaded with FM1-43, which indicates their capacity for repeated vesicular exocytosis and recycling. In contrast, the dye could not as readily be unloaded from UM neuron terminals (type II), which may indicate that they have a slower turnover of synaptic vesicles.

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. In the second part, we show that, in contrast to previous suggestions, DUM neurons are not recruited as a homogeneous population mediating general arousal but differentially, thus forming subpopulations of specific types. The existence or the absence of commonly occurring postsynaptic potentials in paired recordings clearly shows that only specific types of DUM neurons are targeted by the same presynaptic pathways. Within the thoracic ganglia, different subpopulations of DUM neurons can be distinguished by their different local inputs. Furthermore, only specific subpopulations of DUM neurons receive common intersegmental drive and inputs from the subesophageal ganglion. As a result of all our recordings, we propose a scheme for the differential activation of efferent DUM neurons. This scheme is sufficient to explain DUM neuron activity during principal motor programs.

Action of locust neuromodulatory neurons is coupled to specific motor patterns.

1. Many muscles of the locust are supplied by dorsal unpaired median neurons (DUM neurons) that release octopamine and alter the contractions caused by spikes in motor neurons. To determine when these neuromodulatory neurons are normally activated during behaviour, intracellular recordings were made simultaneously from them and from identified motor neurons during the specific motor pattern that underlies kicking. A kick consists of a rapid and powerful extension of the tibia of one or both hind legs that is produced by a defined motor pattern. Only 3 identified DUM neurons of the 20 in the metathoracic ganglion spike during a kick, and they supply muscles involved in generating the kick. Their spikes occur in a distinctive and repeatable pattern that is closely linked to the pattern of spikes in the flexor and extensor tibiae motor neurons. When the extensor and flexor muscles cocontract, these three DUM neurons produce a burst of spikes at frequencies that can rise to 25 Hz, and with the number of spikes (3-15) related to the duration of this phase of the motor pattern. The spikes stop when the flexor muscle is inhibited and therefore before the tibia is extended rapidly. The other DUM neurons which supply muscles that are not directly involved in kicking are either inhibited or spike only sporadically. 2. The activation of a specific subset of DUM neurons during kicking may thus be timed to influence the action of the muscles that participate in this movement and appear to be controlled by the same circuits that determine the actions of the participating motor neurons. These modulatory neurons thus have specific individual actions in the control of movement.

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.

Activity-dependent structural dynamics of insect sensory fibers.

This report analyses the role of neuronal activity in shaping the axonal arborizations of sensory neurons from individually identified filiform hairs on the prosternum of locusts (Locusta migratoria), and their connections with a pair of identified interneurons (A4I1). Afferents from lateral filiform hairs terminate in the ipsilateral neuropil and connect only with the ipsilateral interneuron in all instars. Afferents from ventral filiform hairs possesses ipsi- and contralateral branches and make monosynaptic connections with both interneurons in first instars. In later instars, the ipsilateral branch, and its synaptic connection to the ipsilateral interneuron, is gradually reduced until it is lost in the adult, whereas the contralateral branch, and its synaptic connection with the contralateral interneuron, is strengthened. Therefore, after an initial overgrowth of fibers and synapses, segregation of fibers occurs involving the loss of synaptic connections. This loss of branches and synapses was prevented by immobilizing a subpopulation of ventral and lateral filiform hairs, or each group independently, so that their normal activity was blocked. In such treated animals afferents from ventral filiform hairs retain their ipsi- and contralateral branches until adulthood. We therefore conclude that afferent activity plays an important role in shaping the final structure and connectivity of afferents, as neither the peripheral position of the receptors nor the hormonal environment was changed by these manipulations.

Distribution of input synapses from processes exhibiting GABA- or glutamate-like immunoreactivity onto terminals of prosternal filiform afferents in the locust.

The locust prosternum carries a population of long filiform hairs that are very sensitive to air currents. The sensory afferent neurones that innervate the hairs make strong monosynaptic connections with an identified intersegmental interneurone (A4I1) which is known to contact motor neurones that supply muscles controlling wing angle during flight. In order discover how the synapse between the afferents and interneurone A4I1 might be modulated, the afferents were labelled intracellularly by backfilling with horseradish peroxidase to reveal their central terminals which lie in the prothoracic ganglion. A postembedding immunogold method was used to make a quantitative assessment of the prevalence of immunoreactivity for GABA and glutamate in processes presynaptic to the afferent terminals. In one afferent neurone, where 77 synapses were examined, 40 (52%) of the presynaptic processes were immunoreactive for GABA. When adjacent sections through the same terminal branches were labelled with the two antibodies, it was demonstrated that GABA- and glutamate-like immunoreactivity was present in different populations of presynaptic processes. A series of 110 ultrathin sections was cut through one set of afferent terminal branches and alternate grids were stained with GABA and glutamate antibodies. From these sections, the terminals were reconstructed and the position of 35 input and 21 output synapses mapped. Of the 35 input synapses, 18 (51%) were immunoreactive for GABA, 14 (40%) were immunoreactive for glutamate and 3 (9%) were unlabelled by either antibody. On these terminals, the different classes of input synapses appeared to be intermingled at random with the output synapses made by the afferent, and no pattern governing synapse distribution could be discerned.

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.

Fate of abdominal ventral unpaired median cells during metamorphosis of the hawkmoth, Manduca sexta.

Each of the unfused abdominal ganglia in the larval, pupal, and adult stages of the hawkmoth, Manduca sexta, has two large ventral median neurons with axons that bifurcate to innervate targets on both sides of the abdomen. Although the dendritic structures of the two neurons are similar, their axons branch to innervate distinct sets of target muscles. During metamorphosis both neurons undergo dendritic regression, followed by growth of new arborizations during adult development. The neurons must innervate different targets in the larva and adult, since many larval muscles degenerate and are replaced during metamorphosis. Both neurons were reactive with an antibody to the neuromodulatory compound, octopamine, in the larval and adult stages. Pairwise intracellular recordings in isolated nerve cords revealed spontaneous excitatory synaptic potentials that occurred in the ventral median neurons of each ganglion in an anterior-to-posterior sequence. The synaptic potentials were eliminated when the interganglionic connective was interrupted posterior to the subesophageal ganglion. The ventral median neurons were also excited by tactile stimulation of the body surface in larvae, pupae and adults.

Output connections of a wind sensitive interneurone with motor neurones innervating flight steering muscles in the locust.

The output connections of a bilaterally symmetrical pair of wind-sensitive interneurones (called A4I1) were determined in a non-flying locust (Schistocerca gregaria). Direct inputs from sensory neurones of specific prosternal and head hairs initiate spikes in these interneurones in the prothoracic ganglion. The interneurone with its axon in the right connective makes direct, excitatory connections with the two mesothoracic motor neurones innervating the pleuroaxillary (pleuroalar, M85) muscle of the right forewing, but not with the comparable motor neurones of the left forewing. The connections can evoke motor spikes. The interneurones also exert a powerful, but indirect effect on the homologous metathoracic pleuroaxillary motor neurones (muscle 114), and a weaker, indirect effect on subalar motor neurones of the hindwings. No connections or effects were found with other flight motor neurones, or motor neurones innervating hindleg muscles, including common inhibitor 1 which also innervates the pleuroaxillary muscle. One thoracic interneurone with its cell body in the right half of the mesothoracic ganglion and with its axon projecting ipsilaterally to the metathoracic ganglion receives a direct input from the right A4I1 interneurone. These restricted output connections suggest a role for the A4I1 interneurones in flight steering.

Evidence for octopaminergic nature of peripherally projecting DUM-cells, but not DUM-interneurons in locusts.

In locusts, a median neuroblast in each segmental ganglion gives rise to numerous unpaired progeny--the well known peripherally projecting dorsal-, occasionally ventral-, unpaired median (DUM-, resp. VUM-) neurones together with the lesser known DUM-interneurones 12. We examine the reputed octopaminergic nature of this nerve cell lineage using an anti-octopamine serum recently developed by M. Eckert and J. Rapus 7. This antiserum labels in each segmental ganglion numerous midline neurones, identifiable as DUM- and VUM-cells by their some sizes and positions, projections in DUM-tracts and characteristic T-junctions with bilaterally projecting axons. All octopamine immunoreactive DUM-, and VUM-neurones appear to project to peripheral nerves; their numbers correspond to the number of peripherally projecting DUM- and VUM-neurones identified so far in the examined ganglia. Presumptive DUM-interneurones, i.e. smaller somata interspersed between the peripherally projecting DUM-cells are not octopamine immunoreactive, but, confirming other studies 25, display GABA-like immunoreactivity. We thus suggest, that of the whole DUM-cell population in the examined ganglia, all and only peripherally projecting DUM-neurons are octopaminergic.

Octopamine immunoreactive cell populations in the locust thoracic-abdominal nervous system.

We describe octopamine-immunoreactive somata and their projections in the pro- meso-, meta- and pregenital abdominal-ganglia of locusts. Immunoreactive midline somata were identified as dorsal- and ventral- unpaired median (DUM- and VUM-, respectively) neurones due to their: characteristic large size and positions of somata, primary neurites in DUM-tracts giving rise to T-junctions, and bilaterally projecting axons. In the prothoracic ganglion there are most likely 8 such cells; in the meso- and metathoracic, some 20 each; and in each individual pregenital abdominal ganglion, typically 3. All appear to project to peripheral nerves and their numbers correspond to the number of peripherally projecting DUM-cells identified to date in each ganglion. We suggest that probably all peripherally projecting DUM-cells are octopaminergic in the examined ganglia. Presumptive DUM-interneurones are not octopamine-immunoreactive, but, confirming other studies, are shown to label with an antiserum to gamma-amino butyric acid (GABA). Other octopamine-immunoreactive neurones include a pair of midline, prothoracic, anterior medial cells, not necessarily DUM-cells, and a pair of ventral lateral somata in each thoracic- and the first abdominal ganglion. The latter project intersegmentally in ventral tracts. Intersegmentally projecting octopamine-immunoreactive fibers in dorsal tracts probably arise from a prothoracic DUM-cell, which leaves through suboesophageal nerves, or descending suboesophageal DUM-cells. Thus, the octopamine-immunoreactive system of thoracic and pregenital abdominal ganglia in locust comprises all peripherally projecting DUM-cells and a plurisegmental network.

An identified dorsal unpaired median neurone and bilaterally projecting neurones exhibiting bovine pancreatic polypeptide-like/FMRFamide-like immunoreactivity in abdominal ganglia of the migratory locust.

Three antisera were used to study the distribution and anatomy of bovine pancreatic polypeptide (BPP)-like/FMRFamide-like immunoreactive neurones within the unfused abdominal ganglia of the migratory locust, Locusta migratoria. All the antisera used stained two or more clusters of perikarya, localized anteriorly and posteriorly near the midline within each unfused abdominal ganglion. Double labelling experiments with intracellular dye injection, or differential backfilling, combined with subsequent immunostaining were carried out to identify these neurones. Two of the antisera (antisera 1 and 2, both raised against FMRFamide) stained three groups of midline neurones, located anterior dorsal, anterior ventral and posterior dorsal within the ganglion. Neurones of the former of these two clusters projected via the anterior median nerve to a neurohaemal organ. The posterior cluster of midline cells comprised immunopositive perikarya all but one of which also projected via the anterior median nerve to innervate the neurohaemal organ. Double labelling with Lucifer yellow and antisera 1 and 2 showed that the remaining neurone was the previously identified dorsal unpaired median (DUM)heart 1 neurone. The third antiserum (AK141), also raised against FMRFamide, stained neurones within an anterior dorsal cluster, and in a posterior cluster. Double labelling with differential Co2+/Ni(2+)-backfilling and the antiserum 3 (AK141) demonstrated that the large neurones of both clusters belonged to the population of bilaterally projecting neurones (BPNs), including the DUMheart1 neurone. Since the antisera cross-react with BPP and fail to label neurones when preadsorped with BPP or FMRFamide, we conclude that the labelled neurones contain polypeptides of the FMRFamide/BPP-family.