Connectivity analysis in the thoracic ganglia between local interneurons and DUM neurons
Abstract: Most animals possess a rich repertoire of complex behaviors, which are often regulated and adapted to specific behavioral requirements by neuromodulatory biogenic amines. Octopamine, the invertebrate equivalent of adrenaline/noradrenaline induces, for instances, "fight or flight responses" by increasing the animal’s arousal and switching the whole organism to "dynamic action". Some of the octopaminergic neurons, the efferent dorsal unpaired median (DUM) neurons, are well characterized by neuroanatomical and electrophysiological studies. DUM neurons release octopamine onto peripheral targets and modulate both the contractions of skeletal muscles and the synaptic transmission of motor neurons. Moreover, they mediate changes in metabolism of working muscles. DUM neurons receive subtype specific sensory inputs, and they are activated subtype specifically during different motor behaviors like walking or flying. Interestingly, even though some principles of these pathways are discovered, none of the studies so far was able to detect either presynaptic neurons or monosynaptic sensory input. The aim of my study is to characterize local interneurons that may connect to DUM neurons directly. Via intracellular recording techniques, electrical stimulation, pharmacology and anatomical reconstructions I shall be able to give more insights into modulatory circuits as important components of neuronal networks.
Octopaminergic dorsal unpaired median (DUM) neurons in the desert locust, Schistocerca gregaria Forskål
Locusts possess specialized neurons that release octopamine. Moreover, these cells appear to be efferent paracrine cells (Stevenson et al., 1992; Stevenson and Pflüger, 1994). Octopaminergic cell bodies are located in the midline of all segmental ganglia in the ventral nerve cord except for the brain. They are named with respect to the position of their somata as dorsal unpaired median (DUM), and ventral unpaired median (VUM) neurons. Since their discovery in locusts (Plotnikova, 1969) and cockroaches (Crossman et al., 1971,1972) DUM neurons – especially those in the thoracic ganglia – have been characterized in great detail by many investigations (reviews: Burrows, 1996; Bräunig and Pflüger, 2001; Duch and Pflüger, 2010).
Due to their large somata (20-60 µm) arranged in clusters with a reliable position in the respective thoracic ganglion, DUM neurons are very suitable for intracellular electrophysiological recordings with subsequent dye injection (Bräunig and Pflüger, 2001; Heidel and Pflüger, 2006). Additionally DUM neuron somata are capable of generating overshooting action potentials and can, thus, already be detected while be recorded intracellularly (Crossman et al., 1971).
Originating from their somata, DUM neurons develop a distinct primary neurite that bifurcates bilaterally symmetrical in two main axons. The axon collaterals then run along thoracic nerves to specific targets where some of them form networks of varicose endings from which they release their modulatory substance octopamine (Bräunig et al., 1994; Bräunig, 1997; Bräunig and Eder, 1998; Bräunig and Pflüger, 2001).
Accordingly, the conspicuous branching pattern allows identification of individual DUM neurons after intracellular staining (see Fig.1 and 2; Campbell et al., 1995; Heidel and Pflüger, 2006).
I shall benefit from insects which are ideally suited for investigations in both semi-intact and isolated (completely deafferented) preparations and in which motor patterns can be elicited both mechanically and biochemically. The main advantage of semi-intact preparations is the longevity of recordings, i.e. increased viability. In comparison, isolated preparations do not show artifacts due to movement caused by contractions in muscle tissues, but due to isolation the background activity of cells/neural circuits is usually very low.
Contributing to the fact that central pattern generators (CPGs) in locusts can be activated pharmacologically (fictive movement generation), I will elicit rhythmic motor patterns, e.g. walking and flight, while performing simultaneous recordings from DUM neurons and other unknown neurons that may be connected with them. Therefore, I will employ the muscarinic agonist pilocarpine to induce fictive walking, as well as the biogenic amine octopamine to induce fictive flight. Moreover I will apply different concentration levels of these drugs to evaluate whether there exists a variability in efficacy contributing to different titers, or not.
To identify transmitters involved in the communication between presynaptic interneurons and DUM neurons I will use several antibodies (antiGABA, antiOA, NC82). The antibodies antiGABA and antiOA shall stain selectively synapses containing GABA (inhibitory) and octopamine (excitatory, neuromodulatory), respectively whereas NC82 shall stain the active zones of all present synapses.
Laser Scanning Microscopy + visualization software
I will employ laser-scanning microscopy (LSM) to elucidate the origin of neuronal input to DUM neurons. Therefore intracellularly recorded DUM neurons will be iontophoretically filled with an intracellular marker (neurobiotin) and subsequently treated as whole-mounts with immunohistochemistry (IHC). During IHC, a fluorophor-carrying second antibody (e.g. Cy2+Streptavidin) will be conjugated to neurobiotin. After proper laser light excitation (e.g. Cy2 -> wavelength 488nm) the emission due to fluorescence will be measured with a confocal microscope (Leica, SP2). One scan results in a series of single image stacks each representing a distinct plane of the whole-mount. Finally, the digital whole-mount scans will be exported to visualization software like amira™ or Fiji-Image J for visualization (see Fig.1, 2 and 3), analyzes, and/or remodeling of stained cells.
Due to the fact that neurons with monosynaptic connections must extend in the very vicinity to each other, this tool shall allow me to state, which of the investigated neurons possess direct connections. Furthermore, I shall be able to evaluate the strength of neuronal connections by considering the amount of synapses in areas with overlapping projections. Additionally, three-dimensional reconstruction of neurons and mapping of their position in the respective ganglion shall help to understand the composition of DUM neuron circuitry in detail.
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 Bräunig P, Eder M, 1998, Locust Dorsal Unpaired Median (DUM) Neurones Directly Innervate And Modulate Hindleg Proprioceptors. J. Exp. Biol. 201:3333-3338
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