The Pflüger Research Group

 
 
 Prof. Dr. Hans-Joachim Pflüger

 Freie Universität Berlin 
 Fachbereich Biologie, Chemie, Pharmazie
 Institut für Biologie
 - Neurobiologie -
 Königin-Luise-Str. 28/30 
 14195 Berlin-Dahlem 
 Germany 

 phone:  ++49 30/838-54676 (office)  
         ++49 30/838-56537 (secretary) 
 fax:    ++49 30/838-55455
 e-mail: pflueger@neurobiologie.fu-berlin.de
 

 

Sensory motor and neuromodulatory networks in insects  

Active locomotion is a feature of all animals, and to achieve this animals have developed (i) contractile tissues such as muscles, (ii) sensory receptors that monitor the effects of movements and (iii) networks of neurons in the central nervous system that control them and in addition a are the interface between sensory and motor systems.

We are interested in problems of sensory-motor integration and the subsequent execution of specific motor behaviors. Insects, such as locusts (Locusta migratoria and Schistocerca gregaria) and moths (Manduca sexta), are particularly suited for such studies as they posses a rich  behavioral repertoire. In addition, many of their neurons can be identified, and accessed via sharp electrodes, thus allowing a cellular analysis of behavioral events. 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 octopaminergic system during motor behavior.

 

(1) Development of sensory hairs and their first order interneuron

We 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 interneuron (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 arbors of manipulated versus normal receptors, and (ii) the dendritic tree of the postsynaptic interneuron A4I1. For this we use multiple dye labeling of receptors and neurons and subsequent processing in the scanning confocal microscope. In collaboration with the Obermayer group at the Technical University (Computing) and the Duch group we  use automatic reconstruction algorithms that finally allow us precise morphometric measurements (see project Daniel Münch)

Last but not least, we are interested in the behavioral function of identified circuits which may change between nymphs and adults.

  

(2) Neuromodulation: A new functional role for Octopamine

Our main interest is to study the functional role of a population of octopaminergic neurons in locusts (Locusta migratoria, Schistocerca gregaria) and moths (Manduca sexta). These neurons have some morphological and electrical features that make them unique in the central nervous system: (i) at least some of them are unpaired and therefore posses bilaterally symmetrical axons, (ii) their cell bodies are in a median location either on the dorsal or ventral surface, and hence they are called dorsal or ventral unpaired median, DUM or VUM, neurons, and (iii) they seem to innervate most if not all skeletal and visceral muscles.

In particular, we examine when and during which motor behavior these cells are activated. In contrast to common belief, we identified subpopulations of these cells which are either activated or inhibited during specific motor behaviors. To understand more about these different subpopulations, we have started to examine the various types of DUM neurons in vivo and in vitro.  One goal is to characterize the different types of DUM neurons according to their ionic currents, receptor molecules and signal cascades (whole cell voltage clamp, patch clamp). As an additional step towards this characterization, individually identified DUM neurons are taken into culture and then examined physiologically (see project Dr. Einar Heidel).

One outcome of our previous studies is that during locust flight the DUM-neurons innervating flight muscles are inhibited. This may indiciate that octopamine is downregulated in flight musle. As it has been shown that the octopamine content of flight muscle decreases in flight (Goosey and Candy, Insect Biochem. 12: 681-685, 1982) and that also Fructose-2,6-bisphosphate (F26BP an activator of glycolysis), which is normally stimulated by octopamine, decreases (see Wegener, Experientia 52: 404-412, 1996 and Candy, Becker and Wegener, Comp. Biochem. Physiol. 117B:497-512, 1997), this hints to an additional metabolic role of DUM-neurons. Indeed, we could show that stimulation of identified DUM neurons increases the F26BP content in  the respective target muscles. Further studies to reveal  the intracellular signaling cascades activated by octopamine showed that one of two pathways that are necessary to increase F26BP, involve PKA. In collaboration with Prof. Wegener, Mainz, and PD Dr. Uli Müller, Berlin, we intend to further unravel the intracellular signaling cascades activated by octopamine. 

Based on these results, we therefore suggest octopamine, apart from its role as a modulator of the efficacy of neuromuscular transmission in some systems controls catabolism in muscles. The DUM neurons to wing muscles which are inhibited during flight are active at rest with a low firing frequency. This may lead to accumulation of F26BP in flight muscle which together with AMP will have a boosting effect of glycolytic rate when a high energy demand is required for take-off. However, during sustained flight the respective muscles gain their energy through lipid metabolism, and this may be the reason why all systems that would increase glycolysis, including the DUM neurons to wing muscles, are automatically switched off as soon as the animal opens its wings.

 

Connectivity of octopaminergic DUM and VUM neurons

A remaining unanswered question is that of the connectivity of these neurons. Although we know that DUM/VUM neurons are always activated or inhibited in parallel to motor neurons some important differences exist: (i) the connections of known descending interneurons are not direct, and (ii) no direct sensory input has been identified. Therefore, it seems that all connections to DUM/VUM neurons are through either local interneurons 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 neurons. In addition, we already have shown that all larval DUM/VUM neurons are recruited during fictive crawling behavior, and that  the presynaptic neurons involved in this recruitment reside in the suboesophageal ganglion. Therefore, one project centers on identifying the respective presynaptic neurons.

In addition, we already know that in the adult moth the DUM/VUM neurons 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 connectivity.

 

 

Ultrastructure of neuromodulatory terminals

Finally, a third 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 of type II. From an evolutionary point of view the latter type II most likely represents the original 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. Alan Watson, Cardiff University, UK, and Dr. Natalia Biserova, Moscow State University, Russia.

In this context we are also studying the innervation of visceral muscles, for example the oviduct, in particular with respect to biogenic amines (octopamine) and peptides (allatostatin)  (in collaboration with Dr. Skiebe-Corrette and Jana Börner, Dr. V. Vedenina and Dr. N. Biserova, Moscow. We also look at innervation patterns of the locust heart (in collaboration with Dr. Stevenson, Leipzig).

 

Projects: 

 

Sensory-motor development:

Activity-dependent dynamics of sensory hairs and their first order interneuron A4I1
(Daniel Münch Hyperlink setzen!)

 

Neuromodulatory networks

1) Patch-Clamp recordings from single identified DUM-neurons in cell culture (Dr. Einar Heidel, S. Seiffert)

2) Recruitment of neuromodulatory cells during motor behavior (Hans-Joachim Pflüger) 

3)  Changes in ionic currents of neuromodulatory neurons during metamorphosis (patch clamp, voltage clamp, iontophoresis, pharmacology) (Ricardo Vierck)

4) Functional development of neuromodulatory neurones in a holometabolous insect, Manduca sexta. (In collaboration with Prof. R. B. Levine, Univ. of Arizona, ARL, Division of Neurobiology, Tucson, Arizona, USA.

 

 

Projects for PhD-theses:

 

Neuromodulation

1)   Connectivity of DUM/VUM neurons in insects (intracellular electrophysiology, electrical and sensory stimulation)

2)  Signaling cascades by octopamine in insect muscle (Ca, IP3?) (in collaboration with Dr. U. Müller and Prof. Wegener, Mainz)

 

Projects for students (diploma thesis or Saatsexamen):

 

Development

1) 3D-analysis of neuropiles and tracts in ganglia of locusts, moths and Drosophila (3D-reconstruction with AMIRA, confocal microscopy, immunocytochemistry, neuroanatomy, in collaboration with AG Duch)

2) Development of a receptive field: A scanning electron miscroscopic study in larval and adult locusts (this is a good topic for a Staatsexamen!)

 

Neuromodulation

1) Characterization of descending octopaminergic suboesophageal neurons in Manduca sexta

2) The dendritic morphologies of identified types of actopaminergic DUM neurons in locusts

 

More biochemical studies (in collaboration with other groups)

1) Does octopamine activate the IP3-Ca-signaling pathway in insect muscle? (this project is in collaboration with PD Dr. U. Müller, Berlin)

2) Activation of PFK (phosphofructokinase) by octopamine stimulation (this project is in collaboration with Prof. Wegener, Universität Mainz)

3) Does octopamine influence lipid metabolism in muscle? (this project is in collaboration with Profs. Ferenz and Ziegler, Universität Halle)

 

Methods established in our laboratory

1) extra- and intracellular electrophysiology including neuroanatomical tracing methods (cobalt and fluorescent dyes), patch-clamp, isolated neurons, cell culture

2) methods of behavioral physiology (force and movement measurement)

3) neuroanatomical methods (paraffine and plastic serial sections, kryosections)

4) immunocytochemistry, confocal microscopy including techniques of 3D analysis (in collaboration with AG Duch)

 

FU:N 6/2000 "Reizvolle Einblicke in die Bewegungsmuster kleiner Sechsbeiner"

CV

Publications

Themen für Doktorarbeiten, Diplomarbeiten und Staatsexamensarbeiten

Our latest published manuscripts

Report 2003

GRK 837 "Functional insect science"
siehe unter
http://www.bio.uni-potsdam.de/grkins/index.htm

 

 

last update: October 15, 2004