Sulforhodamine labeling of neural circuits engaged in motor pattern generation in the in vitro turtle brainstem-cerebellum

Abstract

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.

Identification of Muscle Spindles in the Submentalis Muscle of the Marine Toad, Bufo marinus and Its Potential Proprioceptive Capacity in Jaw–Tongue Coordination

“…Several past studies suggest that SR 101, a charged fluorescent molecule, is taken up by endocytosis in synaptically activated neurons (Lichtman et al, 1985;Betz et al, 1992;Keifer et al, 1992;Teng et al, 1999;Anderson, 2001). The ultrastructural studies of muscle spindles in mammals and birds have shown the direct continuity of the perineurium of the entering nerve with the muscle spindles, which consist of collagen (Patten and Ovalle, 1991).…”

In vivo staining of oligodendroglia in the rabbit retina

“…Sulforhodamine 101 was used for studying the activitydependent uptake of the probe in motor endplates (Lichtman et al, 1985) and in a turtle brainstem-cerebellum preparation (Keifer et al, 1992). The studies are all based on the ability of the dye to pass across the cell membrane.…”

In Vivo Labeling of Cortical Astrocytes with Sulforhodamine 101 (SR101)

“…Are reactive astrocytes that typically show reduced gap junction coupling (Xu et al 2009) also labeled by SR101? How can the differences in SR101 staining between rodent cortex and other central nervous system regions or species be reconciled (Keifer et al 1992;Ehinger et al 1994;Lavallee and Pflieger 2009)? Independent of the answers to these questions, SR101 has already proven and will continue to be a valuable tool in the study of astrocyte structure and function in the intact rodent brain and for a number of in vivo applications.…”