Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes.
Membrane-impermeant calcium indicator dyes were used to retrogradely label dorsal root ganglia, spinal motoneurons and interneurons in the spinal cord of the chick embryo. The dyes were also used to label anterogradely primary afferent axons in the spinal cord and synaptic endings in the ciliary ganglion. Labelled neurons were imaged using digital videomicroscopy. Motoneurons and dorsal root ganglion cells exhibited a frequency-dependent change in fluorescence during antidromic stimulation. Single antidromic stimuli resulted in fluorescence transients that could be resolved in individual cells in real time. In addition, fluorescence changes could be recorded in motoneurons during episodes of bursting generated by rhythmic synaptic inputs from premotor networks. Stimulus-induced fluorescence signals were also detected in axons and synaptic endings labelled anterogradely. Optical signals were largely abolished in the absence of extracellular calcium. The results show that calcium changes can now be measured in identified populations of neurons and presynaptic terminals. The strong dependence of these signals on impulse activity suggests that the technique will be useful for monitoring the activity of identified neuronal populations. The calcium-dependent fluorescence signal probably results from cytosolic dye derived from diffusion which may limit the technique to situations in which the dye can be applied close (< 1 cm) to cell bodies.
O'Donovan
M J
MJ
Section on Developmental Neurobiology, NINDS, NIH Bethesda, MD 20892.
Ho
S
S
Sholomenko
G
G
Yee
W
W
D000818:Animals; D001369:Axons / Q000648:ultrastructure; D002118:Calcium / Q000737:chemistry*; D002467:Cell Nucleus / Q000648:ultrastructure; D002642:Chick Embryo; D004396:Coloring Agents; D003593:Cytoplasm / Q000648:ultrastructure; D004558:Electric Stimulation; D005453:Fluorescence; D016257:Fura-2; D005726:Ganglia, Parasympathetic / Q000648:ultrastructure; D005727:Ganglia, Spinal / Q000502:physiology / Q000648:ultrastructure; D007395:Interneurons / Q000502:physiology / Q000648:ultrastructure; D008846:Micromanipulation; D009411:Nerve Endings / Q000648:ultrastructure; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure*; D009475:Neurons, Afferent / Q000648:ultrastructure; D010784:Photomicrography; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D013569:Synapses / Q000648:ultrastructure
D004396:Coloring Agents; D002118:Calcium; D016257:Fura-2