This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Systems/Circuits Command or obey? Homologous neurons differ in hierarchical position for the generation of homologous behaviors Akira Sakurai1 and Paul S. Katz2 1Neuroscience Institute, Georgia State University, Atlanta, GA 30302, USA 2Department of Biology, University of Massachusetts, Amherst, MA 01003, USA https://doi.org/10.1523/JNEUROSCI.3229-18.2019 Received: 22 December 2018 Revised: 20 April 2019 Accepted: 8 May 2019 Published: 17 June 2019 Author contributions: A.S. and P.S.K. designed research; A.S. performed research; A.S. analyzed data; A.S. wrote the first draft of the paper; A.S. and P.S.K. edited the paper; A.S. and P.S.K. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. We thank Dr. Andrey Shilnikov for helpful discussions. We are also grateful to Mr. Trevor Fay and Mr. Andrew Kim at the Monterey Abalone Company for their efforts in collecting and shipping animals to meet our needs. This work was supported by NSF grant IOS-1455527 to PSK. Corresponding author: Akira Sakurai, PO Box 5030, Georgia State University, Atlanta, GA 30302-5030, USA., [email protected] Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.3229-18.2019 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 the authors 1 Title: Command or obey? Homologous neurons differ in hierarchical position for the generation 2 of homologous behaviors. 3 4 Abbreviated title: Command neuron for a half-center oscillator. 5 6 Author names: Akira Sakurai1 and Paul S. Katz2 7 1. Neuroscience Institute, Georgia State University, Atlanta, GA 30302, USA 8 2. Department of Biology, University of Massachusetts, Amherst, MA 01003, USA 9 10 Corresponding author: Akira Sakurai 11 PO Box 5030, Georgia State University, Atlanta, GA 30302-5030, USA. 12 [email protected] 13 14 Number of pages: 36 15 Number of figures: 10 16 Number of words for Abstract: 247 17 Introduction: 643 18 Discussion: 1494 19 Conflict of interest: The authors declare no competing financial interests. 20 21 Acknowledgements: We thank Dr. Andrey Shilnikov for helpful discussions. We are also 22 grateful to Mr. Trevor Fay and Mr. Andrew Kim at the Monterey Abalone Company for their 23 efforts in collecting and shipping animals to meet our needs. This work was supported by NSF 24 grant IOS-1455527 to PSK. 1 25 Abstract (247/250) 26 In motor systems, higher-order neurons provide commands to lower-level central pattern 27 generators (CPGs) that autonomously produce rhythmic motor patterns. Such hierarchical 28 organization is often thought to be inherent in the anatomical position of the neurons. Here, 29 however, we report that a neuron that is member of a CPG in one species acts as a higher-order 30 neuron in another species. In the nudibranch mollusc, Melibe leonina, swim interneuron 1 (Si1) 31 is in the CPG underlying swimming, firing rhythmic bursts of action potentials as part of the 32 swim motor pattern. We found that its homolog in another nudibranch, Dendronotus iris, serves 33 as a neuromodulatory command neuron for the CPG of a homologous swimming behavior. In 34 Dendronotus, Si1 fired irregularly throughout the swim motor pattern. The burst and spike 35 frequencies of Dendronotus swim CPG neurons correlated with Si1 firing frequency. Si1 activity 36 was both necessary and sufficient for the initiation and maintenance of the swim motor pattern. 37 Each Si1 was electrically coupled to all of the CPG neurons and made monosynaptic excitatory 38 synapses with both Si3s. Si1 also bilaterally potentiated the excitatory synapse from Si3 to Si2. 39 “Virtual neuromodulation” of both Si3-to-Si2 synapses using dynamic clamp combined with 40 depolarization of both Si3s mimicked the effects of Si1 stimulation on the swim motor pattern. 41 Thus, in Dendronotus, Si1 is a command neuron that turns on, maintains, and accelerates the 42 motor pattern through synaptic and neuromodulatory actions, thereby differing from its homolog 43 in Melibe in its functional position in the motor hierarchy. 2 44 Significance statement (119/120) 45 Cross-species comparisons of motor system organization can provide fundamental insights into 46 their function and origin. Central pattern generators (CPGs) are lower in the functional hierarchy 47 than the neurons that initiate and modulate their activity. This functional hierarchy is often 48 reflected in neuroanatomical organization. This paper definitively shows that an identified 49 cerebral ganglion neuron that is a member of a CPG underlying swimming in one nudibranch 50 species serves as a command neuron for the same behavior in another species. The authors 51 describe and test the synaptic and neuromodulatory mechanisms by which the command neuron 52 initiates and accelerates rhythmic motor patterns. Thus, the functional position of neurons in a 53 motor hierarchy can shift from one level to another over evolutionary time. 3 54 Introduction (643/650) 55 A hierarchical organization implies directionality, with higher-level components issuing 56 commands that are carried out by lower levels (Kupfermann and Weiss, 1978). This organization 57 is often considered to be inherent in the neuroanatomy of motor systems. For example, brainstem 58 neurons provide commands to locomotor central pattern generators (CPGs) in the spinal cord 59 (Dubuc et al., 2008; Roberts et al., 2008; Kiehn, 2016). Similarly, in invertebrates, commands for 60 locomotion and feeding come from neurons outside of the CPG and often in other ganglia 61 (Gillette et al., 1982; Gamkrelidze et al., 1995; Panchin et al., 1995; Frost and Katz, 1996; Stein, 62 2009; Puhl et al., 2012) . Here, we show that the hierarchical position of a neuron differs in two 63 species even though the neuron is involved in the production of the same behavior in those 64 species. 65 The nudibranch molluscs, Melibe leonina and Dendronotus iris exhibit characteristic 66 swimming behaviors that consist of a series of laterally-directed body flexions in which the 67 whole body alternately bends toward the left and right sides (Sakurai et al., 2011). A 68 phylogenetic analysis indicates that the most recent common ancestor of these species likely 69 swam in this manner, making the swimming behaviors homologous (Goodheart et al., 2015; 70 Sakurai and Katz, 2017). 71 In addition to the behavioral homology, a bilaterally represented neuron was shown to be 72 homologous. First identified in Melibe, the neuron was named Swim Interneuron 1 (Si1) because 73 of its involvement in swim CPG (Thompson and Watson, 2005). It was previously established 74 that Si1 in Dendronotus is homologous to Si1 in Melibe based on its conserved neuronal 75 anatomy (soma location in the cerebral ganglion, proximity to a cluster of serotonergic neurons, 76 unique axon shape) (Fig. 1A1, B1), and neurochemistry (FMRFamide immunoreactivity), which 4 77 uniquely identify this neuron in several nudibranch species (Sakurai et al., 2011; Newcomb et al., 78 2012; Gunaratne et al., 2017). Homologous neurons are a classic feature of gastropod nervous 79 systems (Croll, 1987; Bulloch and Ridgway, 1995; Katz and Quinlan, 2018). 80 In Melibe, Si1 is electrically-coupled to the ipsilateral Swim Interneuron 2 (Si2) and the 81 contralateral Swim Interneuron 4 (Si4) in the pedal ganglia (Fig. 1A2, Sakurai et al, 2014). Each 82 neuron inhibits its contralateral counterpart to form the main half-center oscillator. The burst 83 period of the main half-center oscillator is regulated by inhibitory synapses from Swim 84 Interneuron 3 (Si3) (Fig. 1A2) (Sakurai et al., 2014). Si1 also synapses onto Si3 on both sides, 85 which plays roles in setting the phase relationship and promoting the transition of Si3 bursting 86 from one side to the other (Sakurai et al., 2014). As it is part of the CPG, tonic depolarization or 87 hyperpolarization of one Si1 interferes with rhythmic activity (Fig 1A3,4) (Sakurai et al., 2011). 88 In contrast to Melibe, in Dendronotus, Si1 has no reciprocal inhibition and is not a 89 member of the CPG (Fig. 1B2); each Si1 is electrically coupled to both left and right Si2 and 90 fires irregularly throughout the swim motor pattern (Sakurai et al., 2011; Sakurai and Katz, 2016, 91 2017). The swim CPG is composed of Si2 and Si3, which form the half-center oscillator through 92 reciprocal inhibition of their contralateral counterparts (Fig. 1B2). A homolog of Si4 has not 93 been identified. It was shown that tonic depolarization of one Si1 speeds up bursting, whereas 94 hyperpolarization of Si1 slows it down (Fig. 1B3,4) (Sakurai et al. 2011). However, the 95 mechanism of such modulatory action and its roles in the motor pattern generation were not 96 previously elucidated. 97 Here, we find that unlike its homolog in Melibe, Si1 in Dendronotus is a command 98 neuron for swimming; its spiking activity is necessary for the production of the motor pattern 99 and sufficient to elicit it. Depolarizing Si1 initiates, maintains, and accelerates the swim motor 5 100 pattern through its synaptic and neuromodulatory actions on the members of the swim CPG. 101 Thus, homologous neurons occupy different positions in the motor hierarchies of Dendronotus 102 and Melibe. 103 104 Materials and Methods 105 Animals 106 Adult specimens of Melibe leonina and Dendronotus iris were collected on the west coast of 107 North America by Monterey Abalone Company (Monterey, CA), Marinus Scientific, LLC (Long 108 Beach, CA), and Living Elements Ltd (Delta, BC, Canada). Animals were shipped to Atlanta 109 overnight and kept in artificial seawater (Instant Ocean®, Mentor, OH) at 10 to 12° C with a 110 12:12 light/dark cycle.