CALCIUM IMAGING OF RHYTHMIC NETWORK ACTIVITY IN THE DEVELOPING SPINAL-CORD OF THE CHICK-EMBRYO

Video-rate imaging of spinal neurons loaded with calcium-sensitive dye s was used to investigate the calcium dynamics and cellular organizati on of spontaneously active rhythm-generating networks in the spinal co rd of E9-E12 chick embryos. Spinal neurons were loaded with bath-appli ed fura-2am. Motoneurons were also loaded by retrograde labeling with dextran-conjugated, calcium-sensitive dyes. Dye-filled motoneurons exh ibited large fluorescent changes during antidromic stimulation of moto r nerves, and an increase in the 340/380 fura fluorescence ratio that is indicative of increased intracellular free calcium. Rhythmic fluore scence changes in phase with motoneuron electrical activity were recor ded from motoneurons and interneurons during episodes of evoked or spo ntaneous rhythmic motor activity. Fluorescent responses were present i n the cytosol and in the perinuclear region, during antidromic stimula tion 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 hi ghest proportion in the ventrolateral part of the cord. Fluorescent si gnals were synchronized in different regions of the cord and were simi lar in time course in the lateral motor column and in the intermediate region. In the dorsal region the rhythm was less pronounced and the s ignal 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 interneuro ns exhibited tonic elevations of fluorescence for the duration of the episode whereas others were rhythmically active in phase with motoneur ons. At the onset of each cycle of rhythmic activity the earliest fluo rescent change occurred ventrolaterally, in and around the lateral mot or column, from which it spread to the rest of the cord. The results s uggest that neurons in the ventrolateral part of the spinal cord are i mportant for rhythmogenesis and that axons traveling in the ventrolate ral white matter may be involved in the rhythmic excitation of motoneu rons and interneurons. The widespread synchrony of the rhythmic calciu m transients may reflect the existence of extensive excitatory interco nnections between spinal neurons. The network-driven calcium elevation s in the cytosol and the perinuclear region may be important in mediat ing activity-dependent effects on the development of spinal neurons an d networks.