CALCIUM IMAGING OF MOTONEURON ACTIVITY IN THE EN-BLOC SPINAL-CORD PREPARATION OF THE NEONATAL RAT

1. This paper describes the use of calcium imaging to monitor patterns of activity in neonatal rat motoneurons retrogradely labeled with the calcium-sensitive dye, calcium green-dextran. 2. Pressure ejection of calcium green-dextran into ventral roots and into the surgically peel ed ventrolateral funiculi (VLF) at the lumbar cord labeled spinal moto neurons and interneurons. The back labeled motoneurons often formed tw o to three discrete clusters of cells. 3. Fluorescent changes (10-20%) could be detected in labeled motoneurons after a single antidromic st imulus of the segmental ventral root. These changes progressively incr eased in amplitude during stimulus trains (1-5 s) at frequencies from 5 to 50 Hz, presumably reflecting a frequency-dependent increase in fr ee intracellular calcium. 4. Stimulation of the ipsilateral VLF at the caudal lumbar level (L(6)), elicited frequency-dependent, synapticall y induced motoneuronal discharge. Frequency-dependent fluorescent chan ges could be detected in calcium green-labeled motoneurons during the VLF-induced synaptic activation. 5. The spatial spread of synaptic act ivity among calcium green-labeled clusters of motoneurons could be res olved after dorsal root stimulation. Low-intensity stimulation of the roots produced fluorescence changes restricted to the lateral clusters of motoneurons. With increasing stimulation intensity the fluorescenc e change increased in the lateral cells and could spread into the medi al motoneuronal group. After a single supramaximal stimulus a similar pattern was observed with activity beginning laterally and spreading m edially. 6. Substantial changes in fluorescence of calcium green-label ed motoneurons were also observed during motoneuron bursting induced b y bath application of the glycine receptor antagonist strychnine or th e potassium channel blocker 4-aminopyridine (4-AP). 7. Our results sho w that membrane-impermeant fluorescent calcium indicators can be used as a tool to study the activity of specific populations of spinal neur ons during execution of motor functions in the developing mammalian sp inal cord. They also suggest that lateral clusters of motoneurons in t he developing spinal cord of the rat are more recruitable or excitable than more medial clusters. Further understanding of these findings re quires identification of these clusters.