Calcium imaging of CPG-evoked activity in efferent neurons of the stick insect

Abstract

The stick insect is a well-established experimental animal to study the neural basis of walking. Here, we introduce a preparation that allows combining calcium imaging in efferent neurons with electrophysiological recordings of motor neuron activity in the stick insect thoracic nerve cord. The intracellular free calcium concentration in middle leg retractor coxae motor neurons and modulatory octopaminergic DUM neurons was monitored after backfilling lateral nerve nl5 that contains the axons of these neurons with the calcium indicator Oregon Green BAPTA-1. Rhythmic spike activity in retractor and protractor motor neurons was evoked by pharmacological activation of central pattern generating neuronal networks and recorded extracellularly from lateral nerves. A primary goal of this study was to investigate whether changes in the intracellular free calcium concentration observed in motor neurons during oscillatory activity depend on action potentials. We show that rhythmic spike activity in leg motor neurons induced either pharmacologically or by tactile stimulation of the animal is accompanied by a synchronous modulation in the intracellular free calcium concentration. Calcium oscillations in motor neurons do not appear to depend on calcium influx through voltage-sensitive calcium channels that are gated by action potentials because Calcium oscillations persist after pharmacologically blocking action potentials in the motor neurons. Calcium oscillations were also apparent in the modulatory DUM neurons innervating the same leg muscle. However, the timing of calcium oscillations varied not only between DUM neurons and motor neurons, but also among different DUM neurons. Therefore, we conclude that the motor neurons and the different DUM neurons receive independent central drive.

Mechanosensory stimulation triggers sustained local motor activity inDrosophila melanogaster

“…5). These data resemble the effects of the muscarinic agonist pilocarpine in deafferented insect VNC preparations, which generate rhythmic activity of alternating motor neuron pools controlling joint movement (Baudoux et al, 1998; Chrachri and Clarac, 1987; David et al, 2016; Goldammer et al, 2018; Rillich et al, 2013; Ryckebusch and Laurent, 1993). Moreover, this rhythmic activity is independent within the three leg joints, as these rarely exhibit coordinated activity between them, meaning that different CPGs control the antagonistic activity of motor neuron pools of each leg joint (Bidaye et al, 2018; Büschges et al, 1995; Daun et al, 2019; Ryckebusch and Laurent, 1994).…”

“…These results are similar to the pharmacological stimulation of decapitated flies with stimulatory amines, notably serotonin, dopamine, and octopamine, which lead to locomotor bouts (Yellman et al, 1997). Moreover, studies in deafferented VNCs using the muscarinic cholinergic agonist pilocarpine, have shown alternating and rhythmic activity in antagonistic leg motor neuron pools driven by the CPGs (Bidaye et al, 2018; Büschges et al, 1995; David et al, 2016; Fuchs et al, 2011; Goldammer et al, 2018; Pearson, 1972; Ryckebusch and Laurent, 1993). Despite this large body of work, the identity, location and architecture of the CPG population remains largely elusive.…”

Central pattern generating networks in insect locomotion

“…For the stick insect, it was shown that transection along the ganglionic midline does not impair the pharmacologically induced rhythmicity, implying the existence of hemisegmental CPGs (Büschges et al, 1995). Calcium oscillations accompany the CPG‐driven activity in leg motor neuron pools (Baden & Hedwig, 2009; Goldammer, Mantziaris, Büschges, & Schmidt, 2018), indicating the contribution of calcium transients to the rhythmic activity generated in leg motor neurons. In the stick insect deafferented preparation, after pilocarpine application brief patterns of coordinated activity among motor neuron pools supplying the three main leg joints may spontaneously occur (Büschges et al, 1995).…”