Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo

Abstract

Video-rate imaging of spinal neurons loaded with calcium-sensitive dyes was used to investigate the calcium dynamics and cellular organization of spontaneously active rhythm-generating networks in the spinal cord of E9-E12 chick embryos. Spinal neurons were loaded with bath-applied fura-2am. Motoneurons were also loaded by retrograde labeling with dextran-conjugated, calcium-sensitive dyes. Dye-filled motoneurons exhibited large fluorescent changes during antidromic stimulation of motor nerves, and an increase in the 340/380 fura fluorescence ratio that is indicative of increased intracellular free calcium. Rhythmic fluorescence changes in phase with motoneuron electrical activity were recorded from motoneurons and interneurons during episodes of evoked or spontaneous rhythmic motor activity. Fluorescent responses were present in the cytosol and in the perinuclear region, during antidromic stimulation and network-driven rhythmic activity. Optically active cells were mapped during rhythmic activity, revealing a widespread distribution in the transverse and horizontal planes of the spinal cord with the highest proportion in the ventrolateral part of the cord. Fluorescent signals were synchronized in different regions of the cord and were similar in time course in the lateral motor column and in the intermediate region. In the dorsal region the rhythm was less pronounced and the signal decayed after a large initial transient. Video-rate fluorescent measurements from individual cells confirmed that fluorescent signals were synchronized in interneurons and in motoneurons although the time course of the signal could vary between cells. Some of the interneurons exhibited tonic elevations of fluorescence for the duration of the episode whereas others were rhythmically active in phase with motoneurons. At the onset of each cycle of rhythmic activity the earliest fluorescent change occurred ventrolaterally, in and around the lateral motor column, from which it spread to the rest of the cord. The results suggest that neurons in the ventrolateral part of the spinal cord are important for rhythmogenesis and that axons traveling in the ventrolateral white matter may be involved in the rhythmic excitation of motoneurons and interneurons. The widespread synchrony of the rhythmic calcium transients may reflect the existence of extensive excitatory interconnections between spinal neurons. The network-driven calcium elevations in the cytosol and the perinuclear region may be important in mediating activity-dependent effects on the development of spinal neurons and networks.

Spontaneous Network Activity Transiently Depresses Synaptic Transmission in the Embryonic Chick Spinal Cord

“…The first was the pathway from the V L F onto both motoneurons and interneurons. This was selected because previous work had shown that some of the interneurons projecting into the V L F were active during spontaneous episodes (O'Donovan et al, 1994, O'Donovan andRitter, Wenner, Ho, Whelan, and O'Donovan, unpublished data). The second pathway was the disynaptic, recurrent connection between motoneurons, which may be equivalent to the mammalian Renshaw pathway (Whelan and O'Donovan, 1997;Wenner et al, 1998).…”

Functional Imaging, Spatial Reconstruction, and Biophysical Analysis of a Respiratory Motor Circuit IsolatedIn Vitro

“…A similar approach involving retrograde labeling of neurons, which we call "functional neural tracing," was used here to label XII preMNs and MNs. Our population labeling approach, based on axonal projections and network connectivity, and using acetoxymethyl (AM) forms of Ca 2ϩ -sensitive dyes, is an extension of the methods originally described by O'Donovan et al (1994) and Fetcho and O'Malley (1995) for retrograde labeling of neurons in brainstem-spinal cord motor circuits with Ca 2ϩ -sensitive dyes. Our use of a membranepermeant AM ester form of dye enabled us to retrogradely label neurons with Ca 2ϩ -sensitive dye through axons of passage without affecting axonal or network function (Koshiya and Smith, 1999).…”

GABA: A Pioneer Transmitter That Excites Immature Neurons and Generates Primitive Oscillations

“…In humans, the presence of these patterns is clinically relevant as their absence is thought to reflect a bad prognosis (510). In a series of investigations, O'Donovan and collaborators (190,478,480) reported that the embryonic chick spinal cord has spontaneous activity generated by somewhat hyperexcitable networks that can be recorded already in ovo. They exhibit rhythmic discharge both with slow episodes (lasting minutes) and fast intraepisode cycling (ϳ1 Hz frequency).…”