Journal of Neuroscience, Vol 12, 3187-3199, Copyright © 1992 by Society for Neuroscience
Sulforhodamine labeling of neural circuits engaged in motor pattern generation in the in vitro turtle brainstem-cerebellum
J Keifer, D Vyas and JC Houk
Department of Physiology, Northwestern University Medical School, Chicago, Illinois 60611.
A fluorescent molecular probe was used in combination with a novel in vitro
preparation to study spatial patterns of neural activity associated with
motor pattern generation. The in vitro brainstem- cerebellum preparation
takes advantage of the turtle's unusual resistance to anoxia to preserve
the entire neural network that connects the cerebellum, red nucleus, and
reticular formation. This preparation was bathed in a 0.01% solution of
sulforhodamine while it was activated unilaterally by electrical
stimulation of the dorsal quadrant of the spinal cord for 1 hr.
Sulforhodamine is a small, sulfonated, highly charged fluorescent molecule
that is taken up by endocytosis. To examine its distribution in the
cerebellum and brainstem, coronal sections were prepared and viewed under
epifluorescence illumination. Distinctive spatial patterns of labeling were
associated with unilateral electrical stimulation of the in vitro network,
suggesting that dye uptake was activity dependent. Blockade of uptake with
altered magnesium and calcium concentrations indicated that single spike
discharge evoked ortho- or antidromically was insufficient to induce dye
uptake. Instead, sulforhodamine staining correlated with the presence of
burst discharge that was recorded extracellularly from the red nucleus.
Blockade of burst discharge with excitatory amino acid receptor antagonists
prevented dye uptake in the red nucleus, the lateral cerebellar nucleus,
and other structures that are known to be interconnected by recurrent
anatomical pathways. These results suggest that sulforhodamine is
internalized by intensely active neurons. The spatial distributions of
label support the hypothesis that burst discharges in the turtle red
nucleus are mediated by excitatory amino acid neurotransmitters and
sustained by recurrent excitation in cerebellorubral synaptic pathways.
Positive feedback in these recurrent pathways may provide an important
driving force for the generation of motor programs that control limb
movements.