Active locomotion is a feature of all animals and to achieve movements animals developed (i) muscles which in the case of arthropods are innervated by excitatory motor neurones, inhibitory neurones and neuromodulatory neurones, (ii) possess sensory receptors that monitor the effects of movements and provide information on internal and external stimuli, and (iii) networks of neurones in the central nervous system that are able to generate rhythms (central pattern generators) and, in addition, are the interface for the action of sensory feedback and neuromodulatory systems.
We are interested in problems of sensory-motor integration, the subsequent execution of specific motor behaviours and the functional role of neuromodulation. Insects, such as locusts (migratory locust, Locusta migratoria and desert locust, Schistocerca gregaria) and moths (tobacco hawk moth, Manduca sexta), are particularly suited for such studies as they possess a rich behavioural repertoire. In addition, many of their neurones can be identified and accessed via sharp electrodes, thus allowing a cellular analysis of behavioural events. For example, we studied walking and flight and how the two pattern generators influence each other. Recently, we have shifted some of our main focuses to the development of sensory-motor systems, and to the function of neuromodulatory systems such as the tyraminergic/octopaminergic system during motor behaviour. For example, we examined in the tobacco hawkmoth, Manduca sexta, when during the pupal stage the central pattern generator for flight is built and becomes functional. We have also began to use the fruit fly, Drosophila melanogaster, as a new experimental animal and to examine wildtype, mutant and transgenic flies with respect to tyramine/octopamine signaling and its functional role for behaviour.
We examined the flight behaviour of mutant fruit flies (Drosophila melanogaster) that lack the tyramine-beta-hydroxylase gene and therefore cannot produce octopamine but have increased titres of tyramine. It was shown that flight in the mutant can be elicited, which shows that, at least in the fly, octopamíne is not necessary for inducing flight. However, the flight performance, for example the total flight time, was significantly reduced. This result fully supports our hypothesis of octopamine as a metabolic regulator and modulator of the efficacy of synaptic transmission. In addition, we have accumulated evidence that for motor behaviour it is the ratio between tyramine and octopamine titres that matter (Brembs, B., Christiansen, F., Pflüger, H.-J. & Duch, C. 2007 J. Neurosci. 27 (41):11122-11131).
By using antibodies against tyramine and octopamine we labeled the distribution of the respective neurones in the brain and all segmental ganglia. We found pure tyraminergic neurones predominantly in the brain, and a relatively small number in fused segmental ganglia. Interestingly, we could show that in some tyraminergic neurones octopamine is produced dependent on stress activity or similar behavioural conditions (or on the selected behaviour). (Kononenko, N., Wolfenberg H. and Pflüger, H-J. 2009. J. Comp.Neurol. 512:433-452).
One particular cluster of the lateral deutocerebrum in locusts was found synthesizing octopamine only after the animal experienced stress. The neurons of this OA3/TA-cluster were identified as projection neurones extending to the first four abdominal ganglia and homologous clusters also exist in other insects such as cockroaches (Periplaneta americana, G5-cluster) or the pink-wing stick insect (Sipyloidea sipylus). Thus, we suggest that these neurones are involved in processing stimuli about stress situations to the networks within the thoracic ganglia. (Kononenko, NL, Hartfil, S, Willer, J, Ferch, J Wolfenberg, H,Pflüger, H-J. A population of descending tyraminergic/octopaminergic projection neurons of the insect deutocerebrum. J comp Neurol. 2018, https://doi.org/10.1002/cne.24583)
We also found that tyramine and octopamine have different effects on the flight rhythm generating network in Manduca sexta. Both act as modulators and are not necessary for eliciting flight, but tyramine specifically acts on depressor systems leaving elevator systems unaffected. (Vierk, R., Pflüger, H.-J., Duch, C. J. Comp. Physiol A, 2009. 195:265–277).
The role of different neuromodulators and neurotransmitters in activating motor networks of the thoracic ganglia was further examined in the locust by using neuromodulators such as pilocarpine, tyramine, chlordimeform, or octopamine to elicit fictive motor activity from isolated meso-and metathoracic ganglia in the locust. Both pilocarpine and tyramine elicited concentration dependent fictive stepping and fictive flight patterns simultaneously with only little interactions between the two patterns. In contrast, octopamine and chlordimeform always elicited fictive flight like patterns and even recruited fast motor units of leg muscles to the flight pattern. (Rillich, J, Stevenson, PA, Pflüger, H-J. Flight and Walking in Locusts–Cholinergic Co-Activation, Temporal Coupling and Its Modulation by Biogenic Amines. PLOS One, 2013, 8 (5):1-11, e62899)
In experiments carried out by Daniel Knebel in our lab and in the Ayali-lab in Tel-Aviv we tried to define the “default states” of CPGs. Many motor behaviors, and specifically locomotion, are the product of an intricate interplay between neuronal oscillators known as central pattern generators (CPGs), descending central commands, and sensory feedback loops. The relative contribution of each of these components to the final behavior determines the trade-off between fixed movements and those that are carefully adapted to the environment. We sought to decipher the endogenous, default, motor output of the CPG network controlling the locust legs, in the absence of any sensory or descending influences. We induced rhythmic activity in the leg CPGs in isolated nervous system preparations, using different application procedures of the muscarinic agonist pilocarpine. We found that the three thoracic ganglia, each controlling a pair of legs, have different inherent bilateral coupling. Furthermore, we found that the pharmacological activation of one ganglion is sufficient to induce activity in the other, untreated, ganglia. Each ganglion was thus capable to impart its own bilateral inherent pattern onto the other ganglia via a tight synchrony among the ipsilateral CPGs. By cutting a connective and severing the lateral-longitudinal connections, we were able to uncouple the oscillators’ activity. While the bilateral connections demonstrated a high modularity, the ipsilateral CPGs maintained a strict synchronized activity. These findings suggest that the central infrastructure behind locust walking features both rigid elements, which presumably support the generation of stereotypic orchestrated leg movements, and flexible elements, which might provide the central basis for adaptations to the environment and to higher motor commands. (Knebel, D, Ayali, A.; Pflüger, H-J, Rillich, J. Rigidity and Flexibility: The Central Basis of Inter-Leg Coordination in the Locust. Front. Neural Circuits 10:112. doi: 10.3389/fncir.2016.00112).
Analyzing this data further, the foreleg pattern generators exhibited lateralization. (Knebel, D, Rillich, J., Ayali, A., Pflüger, H-J, Rigosi, E. Ex-vivo recordings reveal desert locust forelimb control is asymmetric. Current Biology 28, R1–R3, November 19, 2018).
This project tries to reveal the function, morphology and ultrastructure of neuromodulatory terminals on target muscles. In the fruit fly (Drosophila melanogaster) motor terminals or neuromuscular junctions between glutamatergic motor neurones and muscles form type I- terminals and those of tyraminerghic/octopaminergic neuromodulatory neurones form type II-terminals. The axons of these neuromodulatory terminals form so-called “beaded fibers” with the axon possessing boutons or varicosities in regular intervals in all studied insects so far. From an evolutionary point of view the type II-terminals most likely represents the original or “more primitive” type of terminals, for example those found on all visceral muscles. (Stocker, B, Bochow, C, Damrau,C, Mathejczyk, T, Wolfenberg,H, Colomb, J, Weber, C, Ramesh, N, Duch, C, Biserova, NM, Sigrist, S, Pflüger, H-J. Structural and Molecular Properties of Insect Type II Motor Axon
Terminals. Front. Syst. Neurosci. 12:5. doi: 10.3389/fnsys.2018.00005).
Due to our limited knowledge on these type II-terminals and the release mechanisms of dense core vesicles we like to study this in the well characterized muscular system of larval and adult fruit flies. This project will be carried out under the supervision of H-J Pflüger and S Sigrist in the Sigrist-laboratory at Freie Universitärt (DFG project, Pf128/35-1) .
According to our results, octopaminergic DUM/VUM neurones are divided into subpopulations that are specifically recruited (activated or inhibited) during motor behaviour, that behave very differently to sensory stimulation and that also exhibit different electrical properties.
In order to study the electrical properties of DUM neurones we developed a method to selectively stain the neurones in-situ with different fluorescent dyes such as Dextran-Tetramethylrhodamine or Dextran-Fluorescine by means of the retrograde axonal diffusion technique. This allowed unequivocal identification of the type of a DUM neurone according to its colour code, and also allowed in situ recordings from individually labeled DUM neurones with sharp electrodes.
To characterize the ionic currents the whole DUM neurone cluster was removed from the ganglion, individual somata isolated and cultured for up to 24 hours. As the isolated neurones kept their fluorescence they could be easily characterized as belonging to a particular type. Their ionic currents were then examined by the whole-cell-patch clamp configuration.
Only under these conditions it was possible to study quantitative differences, for example in the densities of particular ion channels, and compare in situ- and in vivo- patch-clamp recordings. The most prominent feature of octopaminergic DUM neurones is the generation of action potentials in the soma (active soma spikes), a unique feature neither found in motor neurones nor interneurones. Na+-, Ca2+-, and K+-currents as well as a hyperpolarization-activated current (Ih) were thus characterized with respect to their activation/inactivation properties as well as current densities. In addition, a Ca2+-activated K+-current (IKCa) was described that could be blocked by Cadmium and Charybdotoxine. The overshooting soma-action potentials are carried by sodium and calcium, wheras repolarisation is caused by K+-currents, in particular by a transient A-current which largely controls the firing frequency. An opposing hyperpolarization-activated current (Ih) contributes to maintaining the resting potential and induces “rebound-behavior” after phases of inhibition. Interestingly, the different types of DUM neurones possess different current densities which correspond nicely to the electrical (firing) properties of these neurones described in situ. For example, DUM3 neurones posess more Ik and less INa or Ih , whereas the easily excitable DUM3,4,5 neurones and the DUMETi neurone possess less Ik and more Ih . These results are already published (Heidel E., Pflüger, H.-J. 2006 European Journal of Neuroscience 23: 1189-1206).
To further characterize these neurones we also studied calcium-signaling in the somata and found a new voltage-dependent signaling pathway in these neurones (Ryglewski, S.; Pflüger, H.-J.; Duch, C. 2007.PLoS Biology 5(4): 818 – 827). These studies are now extended to record optophysiologically by calcium-sensitive fluorescent dyes from the whole population of thoracic DUM-neurons during motor activity in the fruit fly (Drosophila melanogaster, project of Dr. Marco Schubert).
Last but not least, we are interested in the behavioural function of identified circuits which may change during development of hemi- or holometabolous insects and during evolution. They may be conserved in different species, or they may have undergone interesting adaptive changes. Therefore, we are also interested in a comparative approach to study well characterized circuits and networks.
As mentioned before, we plan to study the tyraminergic and octopaminergic systems in larval and adult flies, and how these two biogenic amines contribute to controlling motor behaviour. By generating transgenic Drosophila in which all tyraminergic and octopaminergic neurones are labeled by GFP, we can trace the functional changes occurring during metamorphosis where all tyraminergic/Octopaminergic neurons persist but re-innervate newly formed adult muscles. In particular, we are interested in the contribution of these neuromodulatory terminals to development of adult structures and, thus, will study these neurones including their axon terminals in larval, pupal and adult stages. Another interest is concerned with the release mechanisms in aminergic terminals and for this we also collaborate with the Sigrist group at Freie Universität and the group of David Owald at Charité. International collaborators are Natalia M. Biserova, Moscow, Russia; Alex Koon, Hongkong, China and Leena Thorat, Pune, India.
Project on Drosophila type II terminals (N.N.), DFG PF 128/35-7