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Section on Developmental Neurobiology, Laboratory of Neural Control, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland
Submitted 15 February 2005; accepted in final form 24 September 2005
Intracellular Cl– ([Cl–]in) homeostasis is thought to be an important regulator of spontaneous activity in the spinal cord of the chick embryo. We investigated this idea by visualizing the variations of [Cl–]in in motoneurons retrogradely labeled with the Cl-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ) applied to cut muscle nerves in the isolated E10–E12 spinal cord. This labeling procedure obviated the need for synthesizing the reduced, cell-permeable dihydro-MEQ (DiH-MEQ). The specificity of motoneuron labeling was confirmed using retrograde co-labeling with Texas Red Dextran and immunocytochemistry for choline acetyltransferase (ChAT). In MEQ-labeled motoneurons, the GABAA receptor agonist isoguvacine (100 µM) increased somatic and dendritic fluorescence by 7.4 and 16.7%, respectively. The time course of this fluorescence change mirrored that of the depolarization recorded from the axons of the labeled motoneurons. Blockade of the inward Na+/K–/2Cl– co-transporter (NKCC1) with bumetanide (20 µM) or with a low-Na+ bath solution (12 mM), increased MEQ fluorescence by 5.3 and 11.4%, respectively, consistent with a decrease of [Cl–]in. After spontaneous episodes of activity, MEQ fluorescence increased and then declined to the pre-episode level during the interepisode interval. The largest fluorescence changes occurred over motoneuron dendrites (19.7%) with significantly smaller changes (5.2%) over somata. Collectively, these results show that retrogradely loaded MEQ can be used to detect [Cl–]in in motoneurons, that the bumetanide-sensitive NKCC1 co-transporter is at least partially responsible for the elevated [Cl–]in of developing motoneurons, and that dendritic [Cl–]in decreases during spontaneous episodes and recovers during the inter-episode interval, presumably due to the action of NKCC1.
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