Abstract
Locomotor systems are widely used to study rhythmically active neural networks. These networks have to be coordinated in order to produce meaningful behavior. The crayfish swimmeret system is well suited to investigate such coordination of distributed neural oscillators because the neurons and their connectivity for generating and especially for coordinating the motor output are identified. The system maintains a fixed phase lag between the segmental oscillators, independent of cycle period. To further the understanding of the system’s plasticity for keeping the phase lag fixed, we profiled the neurotransmitters used by the Coordinating Neurons, which are necessary and sufficient for coordination of the segmental oscillators. We used a combination of electrophysiological, immunohistochemical, and mass spectrometric methods. This arrangement of methods ensured that we could screen for several specific neurotransmitters, since a single method is often not suitable for all neurotransmitters of interest. In a first step, to preselect neurotransmitter candidates, we investigated the effect of substances known to be present in some swimmeret system neurons on the motor output and coordination. Subsequently, we demonstrated electrophysiologically that the identified synapse between the Coordinating Neurons and their target is mainly chemical, but neither glutamate antagonist nor γ-aminobutyric acid antagonist application affected this synapse. With immunohistochemical experiments, we provide strong evidence that the Coordinating Neurons are not serotonergic. Single-cell MALDI-TOF mass spectrometry with subsequent principal component analysis identified acetylcholine as the putative neurotransmitter for both types of Coordinating Neurons.
Highlights
Animals were anesthetized in ice, exsanguinated, decapitated, and the nervous system carefully dissected from thoracic ganglion 4 (T4) to the terminal abdominal ganglion (A6)
Coordinating information is integrated by one neuron in each hemisegment: Each spike from the Coordinating Neurons elicits a distinct excitatory postsynaptic potentials (EPSPs) in their target ComInt 1, which, in turn, is electrically coupled to one of the segment’s central pattern generators (CPGs) neurons [14, 15]
We first identified neurotransmitters and neuromodulators that changed the swimmeret system’s motor activity, the phase relationship between segments, because they could possibly act by influencing the information transmission between the Coordinating Neurons and ComInt 1
Summary
All experiments were carried out in isolated abdominal nervous systems of the signal crayfish Pacifastacus leniusculus, DANA 1852, of both sexes. For the blocking experiments we averaged and compared amplitudes and spike indices of at least 10 EPSPs under control condition, and at the beginning and end of DNQX and PTX application using custom-written MATLAB (MathWorks, Natick, MA, USA) scripts. Under a stereo fluorescence microscope (SteREO Lumar V12, Zeiss) equipped with a Lumar 43 HE Cy3 filter, the soma was isolated and dissected from the ganglion using ultra-fine scissors, and transferred manually with a glass capillary to a stainless steel sample plate for matrix assisted laser desorption/ionization (MALDI)—time of flight (TOF) mass spectrometry. To compare MALDI-TOF data of single ASCE, DSC, and motor neuron preparations, we used ClinPro Tools software 3.0 (Bruker Daltonics) for comparison of signal intensities in a mass range of m/z 100–300. MALDI MS data were uploaded and the following settings for PCA analysis were used: resolution 800, top hat baseline at 10.0% minimal baseline width, enable smoothing at 0.1 width (m/z) with 5 cycles using Savitzky-Golay filter type, recalibration of 1000 ppm maximal peak shift, 30% match to calibrant peaks, and a peak picking on total average spectrum at 4.00 signal to noise threshold
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