Animal locomotion involves the coordination of multiple muscles under the control of the nervous system. To ensure smooth movements, diverse regulatory mechanisms operate at different hierarchical levels within the motor system, narrowing down the degrees of freedom of a highly distributed system with multiple units. The overall functional organization and operational logic of motor systems, comprising the components at all levels of the hierarchy and their feedforward and feedback interactions, remain, to a great extent, an open question in both vertebrates and invertebrates (Kiehn, 2016; Arber and Costa, 2018; Barkan and Zornik, 2019; Grätsch et al., 2019); this includes the mechanisms that operate at the level of the nerve cord (Grillner and El Manira, 2020; McLean and Dougherty, 2015; Gal et al., 2017; Sengupta and Bagnall, 2023; Calabrese and Marder, 2025). Analysis of this organization across different organisms, developmental stages, and behavioral repertoires has provided fruitful information. Leeches have been highly useful to study motor control due to their robust repertoire of motor behaviors, which are executed by a relatively simple body plan and controlled by a similarly simple nervous system. This system is composed of a chain of 21 midbody ganglia flanked by head and tail brains (Wagenaar, 2015), where each midbody ganglion contains the sensory and motor neurons that innervate the corresponding segment (Muller et al., 1981).
On solid surfaces, leeches exhibit a robust rhythmic motor pattern known as crawling (Figure 1A). This behavior results from waves of elongation and contraction of the body along its antero-posterior axis, as the animal is anchored on the posterior and anterior suckers, respectively (Stern-Tomlinson et al., 1986, Figure 1A). Fictive crawling (crawling) can be monitored in the isolated nervous system, where identified interneurons and motoneurons can be recorded intracellularly and extracellularly (Baader, 1997; Baader and Kristan, 1992; Eisenhart et al., 2000). The pattern is characterized by the alternating activation of the motoneurons, such as CV and DE-3, that innervate circular and longitudinal muscles, respectively (Figure 1B). This pattern can be readily evoked by dopamine in the isolated nerve cords (Puhl and Mesce, 2008), in short chains of ganglia (Kearney et al., 2022), and in single isolated ganglia (Puhl and Mesce, 2008; Rodriguez et al., 2012). These studies established that each ganglion contains the network responsible for producing the rhythmic motoneuron activity compatible with crawling. Note that in the context of this article fictive crawling refers to the rhythm generation but does not include the connectivity that rules intersegmental interactions and the consequent intersegmental lags.
The crawling motor pattern.
(A) Schematic description of a leech crawling step that results from coordinated waves of elongation (i–ii) and contraction (iii–iv) phases, anchored on front and rear suckers. (B) Intracellular recording of CV and extracellular recording of DE-3 (in DP nerve) motoneurons during a dopamine-induced crawling episode in an isolated midbody ganglion. Recording diagram on the left. (C) Putative neuronal circuitry underlying crawling and the recurrent inhibitory circuit in a midbody ganglion. Units C and E correspond to contraction and elongation units of the segmental oscillator, respectively. DE-3 and CV are examples of motoneurons involved in each phase. NS neuron is connected to DE-3 and CV (Rela and Szczupak, 2003; Rodriguez et al., 2009) through chemical and electrical synapses; the + and –symbols indicate the polarity at which the rectifying synapse conducts.
Studies on motor behaviors in diverse organisms show that, at the final stage of neural processing, motoneuron activity is controlled by multiple signal sources (El Manira, 2023; Zhen and Samuel, 2015; Rotstein et al., 2017). Analysis of the nature and effects of these signals on the motor pattern is an active field of research. The present work has focused on the role played by nonspiking (NS) premotor neurons on leech crawling. These neurons are present as bilateral pairs in each segmental ganglion and are functionally analogous to mammalian Renshaw cells (Szczupak, 2014). These spinal cord cells deliver inhibitory signals to the motoneurons in proportion to the motoneuron activity, forming an activity-dependent feedback mechanism that regulates motoneuron output (Alvarez and Fyffe, 2007).
Each pair of NS neurons is at the center of a recurrent inhibitory circuit mediated by chemical and electrical synaptic connections with the motoneurons (Figure 1C; Rela and Szczupak, 2003; Rodriguez et al., 2009). In the context of this circuit, the activity of excitatory motoneurons evokes chemically mediated inhibitory synaptic potentials in NS. Additionally, the NS neurons are electrically coupled to virtually all excitatory motoneurons via rectifying junctions that conduct when the transjunction NS-motoneuron potential is negative. In physiological conditions, this coupling favors the transmission of inhibitory signals from NS to motoneurons. Given this recurrent inhibitory circuit, NS premotor neurons could operate as modulators of motoneuron activity. A previous study (Rodriguez et al., 2012) indicated that NS membrane potential oscillates in tune with the crawling motor pattern and that this premotor neuron can indeed modulate motor activity. In that study crawling was monitored by the recording of a particular motoneuron, the dorsal excitor cell 3 (DE-3). However, the wide connectivity of NS with several excitatory motoneurons that fire during crawling called for a more comprehensive readout of the motor pattern.
The present study aimed to evaluate the influence of the premotor NS neuron across the different stages of crawling. For this purpose, we describe the pattern of activity of different neurons during crawling that were simultaneously monitored through extracellular nerve recordings while manipulating the NS membrane potential. The study reveals that the premotor neuron mediated a reduction of the firing frequency of motoneurons recruited during a specific phase of crawling.