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

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 GABA_A 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.