Behaviors are a product of underlying neural circuits, yet there is a paucity of mechanistic information about how nervous systems contribute to the repeated evolution of similar behaviors. Theoretical studies have predicted that the same behavioral output can be generated by neural circuits with different properties. Here, we test the theory in biological circuits by comparing the central pattern generator (CPG) circuits underlying swimming behaviors in nudibranchs (Mollusca, Gastropoda, Euthyneura, Nudipleura). In comparative studies of neural circuits, neurotransmitter content can serve as landmarks or molecular markers for neuron types. Here, we created a comprehensive map of GABA-immunoreactive neurons in six Nudipleura species. None of the known swim CPG neurons were GABA-ir, but they were located next to identifiable GABA-ir neurons/clusters. Despite strong conservation of the GABA-ergic system, there were differences, particularly in the buccal ganglia, which may represent adaptive changes. We applied our knowledge of neurotransmitter distribution along with morphological traits to identify the neuron type Si1 in Flabellina, a species that swims via whole body left-right (LR) flexions and in Tritonia, a dorsal-ventral (DV) swimming species. Si1 is a CPG member of the LR species Melibe, whereas its homologue in the LR species Dendronotus is not. In Flabellina, Si1 was part of the LR CPG and despite having similar synaptic connections as Flabellina and Melibe, Si1 in Tritonia was not part of its DV swim CPG. Side by side circuit comparison of Flabellina, Melibe and Dendronotus revealed different combinations of circuit architecture and modulation resulting in different circuit configurations for LR swimming. This includes differences in the role and activity pattern of Si1, sensitivity to curare and the effect of homologues of C2, a DV CPG neuron, on the LR motor pattern. These results collectively reveal three different circuit variations for generating the same behavior. It suggests that the neural substrate from which behaviors arise is phylogenetically constrained. While this neural substrate can be configured in multiple different ways to generate the same outcome, the possibilities are finite and, as seen here, similar structural and functional neural motifs are used in the evolution of these circuits.

These studies compare neurotransmitter localization and the behavioral functions of homologous neurons in nudibranch molluscs to determine the types of changes that might underlie the evolution of species-specific behaviors. Serotonin (5-HT) immunohistochemistry in eleven nudibranch species indicated that certain groups of 5 HT-immunoreactive neurons, such as the Cerebral Serotonergic Posterior (CeSP) cluster, are present in all species. However, the locations and numbers of many other 5 HT-immunoreactive neurons were variable. Thus, particular parts of the serotonergic system have changed during the evolution of nudibranchs. To test whether the functions of homologous neurons are phylogenetically variable, comparisons were made in species with divergent behaviors. In Tritonia diomedea, which crawls and also swims via dorsal-ventral body flexions, the CeSP cluster includes the Dorsal Swim Interneurons (DSIs). It was previously shown that the DSIs are members of the swim central pattern generator (CPG); they are rhythmically active during swimming and, along with their neurotransmitter 5-HT, are necessary and sufficient for swimming. It was also known that the DSIs excite efferent neurons used in crawling. DSI homologues, the CeSP-A neurons, were identified in six species that do not exhibit dorsal-ventral swimming. Many physiological characteristics, including excitation of putative crawling neurons were conserved, but the CeSP A neurons were not rhythmically active in any of the six species. In the lateral flexion swimmer, Melibe leonina, the CeSP-A neurons and 5-HT, were sufficient, but not necessary, for swimming. Thus, homologous neurons, and their neurotransmitter, have functionally diverged in species with different behaviors. Homologous neurons in species with similar behaviors also exhibited functional divergence. Like Melibe, Dendronotus iris is a lateral flexion swimmer. Swim interneuron 1 (Si1) is in the Melibe swim CPG. However, its putative homologue in Dendronotus, the Cerebral Posterior ipsilateral Pedal (CPiP) neuron, was not rhythmically active during swim-like motor patterns, but could initiate such a motor pattern. Together, these studies suggest that neurons have changed their functional relationships to neural circuits during the evolution of species-specific behaviors and have functionally diverged even in species that exhibit similar behaviors.