Chloride-Sensitive MEQ Fluorescence in Chick Embryo Motoneurons Following Manipulations of Chloride and During Spontaneous Network Activity

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Abstract
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.