Chloride-sensitive MEQ fluorescence in chick embryo motoneurons following manipulations of chloride and during spontaneous network activity

Chloride-sensitive MEQ fluorescence in chick embryo motoneurons following manipulations of chloride and during spontaneous network activity. J Neurophysiol 95: 323-330, 2006. First published September 28, 2005; doi: 10.1152/jn.00162.2005. 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 GABA(A) receptor agonist isoguvacine (100 mu M) 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 mu M) 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.