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

Jens Goldammer,Charalampos Mantziaris,Ansgar Büschges,Joachim Schmidt,Gennady Cymbalyuk

doi:10.1371/journal.pone.0202822

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.

Highlights

Two approaches allow recording of voltage changes across neuronal membranes: electrodebased techniques and optical measurements
Mesothoracic leg nerves nl5 were backfilled with the Ca2+ indicator OGB-1 to study in situ the free intracellular Ca2+ concentration ([Ca2+]i) in retractor coxae motor neurons and neuromodulatory dorsal unpaired median (DUM) neurons that were stimulated pharmacologically [41] or by stimulation of mechanosensors on the animals body [56]
We show that rhythmic activity in leg motor neurons induced by pharmacological activation of central pattern generators (CPGs) or switching spike burst acitivity in antagonistic motor neurons evoked by tactile stimulation of the experimental animal is accompanied by a synchronous modulation in [Ca2+]i leg motor neurons as monitored by Oregon Green BAPTA-1 that was retrogradely filled into the motor neurons

Summary

Introduction

Two approaches allow recording of voltage changes across neuronal membranes: electrodebased techniques and optical measurements. An advantage of optical measurements over electrode-based techniques is that it is much easier to simultaneously measure activity in several neurons or at different locations of the same neuron. Calcium imaging in stick insect efferent neurons

Results

Discussion

Conclusion