Diffuse distribution of sulforhodamine-labeled neurons during serotonin-evoked locomotion in the neonatal rat thoracolumbar spinal cord.

The fluorescent dye sulforhodamine-101 undergoes synaptic activity-dependent endocytotic uptake and consequent retrograde transport in presynaptic neurons. We used sulforhodamine to identify thoracolumbar spinal premotor neurons (T11-L6) activated during serotonin (5-HT) -induced hindlimb locomotor-like activity in the in vitro neonatal rat spinal cord preparation. Sulforhodamine labeling required locomotor-like activity because few neurons were labeled unless bath applied 5-HT recruited the locomotor rhythm. In contrast, N-methyl-D-aspartate (NMDA; 5 microM) profoundly increased spinal neuronal labeling irrespective of locomotor activity. The contribution of false-positive activity labeling during locomotion induced by application of NMDA with 5-HT (Kjaerulff et al. [1994] J Physiol (Lond). 478:265-273) necessitated the present re-mapping of sulforhodamine-labeled neurons. During 5-HT-evoked locomotion, the sulforhodamine-labeled neurons were diffusely scattered within the spinal cord with predominant labeling in lamina VII. Motor nuclei (lamina IX) and superficial laminae (I-II) were typically devoid of labeled cells in the isolated spinal cord. However, unilateral labeling of motoneurons was achieved when the ipsilateral hindlimb remained attached, suggesting that uptake in motoneurons requires an intact neuromuscular junction. The rostrocaudal incidence and distribution of labeled neurons was uniform in spinal segments L1-L5, with reduced numbers observed in thoracic and L6 spinal segments. Mean total cell labeling was less than 400 per spinal segment, suggesting recruitment from a very small fraction of the neurons contained within the spinal cord (calculated at < 0.1%). These results are consistent with the limited transfer of locomotor-related synaptic activity (Raastad et al. [1996] Neuron 17:729-738) and severe synaptic fatigue (Lev-Tov and Pinco [1992] J Physiol. 447:149-169; Pinco and Lev-Tov [1993] J Neurophysiol. 70:1151-1158; Fleoter and Lev-Tov [1993] J Neurophysiol. 70:2241-2250) observed in the neonatal rat spinal cord. Copyright 2000 Wiley-Liss, Inc.