The Pflüger Research Group
Neuromodulatory and sensory-motor networks in insects
Active locomotion is a feature of all animals, and to achieve this animals have developed (i) muscles which are innervated by excitatory motor neurones, inhibitory neurones and neuromodulatory neurones, (ii) sensory receptors that monitor the effects of movements, and (iii) networks of neurones in the central nervous system that are able to generate rhythms (central pattern generator) and, in addition, are the interface for the action of sensory feedback and neuromodulatory systems.
We are interested in problems of sensory-motor integration and the subsequent execution of specific motor behaviours. Insects, such as locusts (Locusta migratoria and Schistocerca gregaria) and moths (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 study 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 became functional.We have also begun to use the fruit fly, Drosophila melanogaster, as a new experimental animal and examine wildtype, mutant and transgenic flies with respect to tyramine/octopamine signaling and its functional role for behavior.
The flight performance of mutant Drosophila that lack octopamine
Recently, in collaboration with Prof. Dr. Carsten Duch (Tempe, Arizona, USA) and PD Dr. Björn Brembs, Berlin, Frauke Christiansen carried out her Diploma thesis on mutant Drosophila 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 eliciting 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. In addition, we have accumulated evidence that for motor behaviour it is the ratio between the tyramine and octopamine titres that matter (Brembs, B., Christiansen, F., Pflüger, H.-J. & Duch, C. 2007 J. Neurosci. 27 (41):11122-11131).
In the future 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. Thus, in collaboration with the Duch-group in Tempe, AZ, USA and the Sigrist-group at the FUB we plan to generate transgenic Drosophila in which all tyraminergic and octopaminergic neurones are labeled by GFP. 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.
Tyramine is a precursor of octopamine and an independent transmitter.
Recently we used antibodies against tyramine and octopamine to label the distribution of the respective neurones in the brain and all segmental ganglia, We found pure tyraminergic neurones predominantly in the brain, and a relative small number in fused segmental ganglia. Interestingly, we could show that in some tyraminergic neurones octopamine is produced dependent on activity or behavioural conditions (or on the selected behaviour). (Kononenko, N., Wolfenberg H. and Pflüger, H-J. 2009. J. Comp. Neurol. 512:433-452).
In the future we would like to extend these immunohistochemical studies to Drosophila.
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 (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 is further examined by Dr. Jan Rillich.
Connectivity of octopaminergic DUM and VUM neurons
A remaining unanswered question is that of the connectivity of these neurones. Although we know that DUM/VUM neurones are always activated or inhibited in parallel to motor neurones some important differences exist: (i) the connections of known descending interneurones are not direct, and (ii) no direct sensory input has been identified. Therefore, it seems that all connections to DUM/VUM neurones are through either local interneurones or by yet unknown descending neurons from the suboesophageal ganglion.
Momentarily, we study their connectivity in the moth Manduca sexta as it has some advantages over the locust. First of all it has less DUM or VUM neurones. In addition, we already have shown that all larval DUM/VUM neurones are recruited during fictive crawling behaviour, and that the presynaptic neurones involved in this recruitment reside in the suboesophageal ganglion. Therefore, one project centers on identifying the respective presynaptic neurones. In this DFG-funded project we closely collaborate with Prof. Peter Bräunig, RWTH Aachen.
In addition, we already know that in the adult moth the DUM/VUM neurones persist, but after metamorphosis should reveal a totally different recruitment scheme as now some of them innervate wing muscle and the others leg muscle. Therefore, another project looks into the developmental changes of the flight system and when, for example, the motor circuits for flight become functional (Vierk, R., Duch, C., Pflüger, H-J. 2010. J. Comp. Physiol. A 196:37-50).
Ultrastructure of neuromodulatory terminals
This project tries to reveal the ultrastructure of neuromodulatory terminals on target muscles. Normal motor terminals are of type I morphology (in Drosophila terminology), and neuromodulatory terminals are of type II. From an evolutionary point of view the latter type II most likely represents the original or “more primitive” type of terminals, for example those found on all visceral muscles. At present, our knowledge on these types of terminals is rather limited, and thus we like to study this in our well characterized insect muscular system. These studies are carried out in collaboration with Dr. Natalia Biserova, Moscow State University, Russia.
In this context we also studied the innervation of visceral muscles, for example the oviduct, in particular with respect to biogenic amines (octopamine) and peptides (allatostatin). We also look at innervation patterns of the locust heart.
Electrical and cellular properties of identified DUM neurons and optophysiological recordings
According to our results octopaminergic DUM/VUM neurones are divided into subpopulations that are specifically recruited (activated or inhibited) during motor behavior, 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 motorneurones 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 of these neurones 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 (project Dr. Marco Schubert).
Development of sensory hairs and their first order interneuron
We also examine the postembryonic changes of a sensory-motor circuit in locusts which is used for flight steering in the adult animal. Detailed studies are concerned with the organisation of the receptive field of an identified ventral cord interneurone (A4I1), and how hormones and activity dependent processes are involved in this development. We also study the A4I1-interneurone and its connectivity in nymphs and adults, in particular the changes of synaptic transmission that may occur during postembryonic development.
As we can identify many of the receptors, this allows us to manipulate particular receptors, for example block their normal neuronal activity, and study the effects with respects to (i) the axonal arbours of manipulated versus normal receptors, and (ii) the dendritic tree of the postsynaptic interneurone A4I1. For this we use multiple dye labeling of receptors and neurones and subsequent processing in the scanning confocal microscope. Recently, we used this system to study the effects of NO on the filiform hair to interneuron synapses. It was shown by Na-diaphorase staining and by using a universal NOS-antibody that mechanosensory neuropiles in which the above mentioned synapses are situated, are densely labeled by immunoreactivity (Münch, D,, Ott, S., Pflüger, H.-J. , 2010, J. Comp. Neurol. 518:2903-2916). In his PhD-thesis Daniel Münch has also found other, very interesting interneurones that receive inputs from these prothoracic filiform hairs, and we plan to study their behavioural function in more detail.
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 very 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. In collaboration with Prof. Field, Christchurch, New Zealand, we look at very ancient insects such as the Wetas which still show original features. In fact we found that the octopaminergic system is very conserved and many features can already be observed in these insects. In addition, in collaboration with Dr. Georg Mayer, Jena, we study Onychophorans which may be close to arthropods (Meyer et al. 2010, BMC Evol. Biol. 10:255).
Group members' current projects:
(1) Descending suboesophageal-neurones and motor behaviour im Manduca sexta (Jessica Erdmann, PhD-thesis)
(2) The identification of descending tyraminergic/octopaminergic projection neurons of the locust brain and ventral nerve cord (Sergej Hartfil)
(3) Tyraminergic/Octopaminergic neurones in Drosophila: their distribution, their targets and their function (Pflüger and PhD thesis Konstantin Lehmann, and collaboration with Duch, Sigrist, Brembs, Colomb)
(4) The role of neuromodulators in motor pattern generation (see Dr. Jan Rillich, Konstantin Lehmann, Leonard Nadler and Jan Untiedt, Diploma theses)
Methods established in our laboratory
FU:N 6/2000 "Reizvolle Einblicke in die Bewegungsmuster kleiner Sechsbeiner"Themen für Masterarbeiten, Bachelorarbeiten / Topics for student theses Scans of the thoracic gangliaGRK 837 "Functional insect science"
siehe unter http://www.stud.uni-potsdam.de/~grk837/