Profiling neurotransmitters in a crustacean neural circuit for locomotion.

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.

The swimmeret system of crayfish: a practical guide for the dissection of the nerve cord and extracellular recordings of the motor pattern.

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity . The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo. The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students' practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish's abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system. Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.