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J Neurophysiol (September 28, 2005). doi:10.1152/jn.00162.2005
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Submitted on February 15, 2005
Accepted on September 24, 2005

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

Nikolai Chub1*, George Z. Mentis1, and Michael J. O'Donovan1

1 Laboratory of Neural Control, NINDS/NIH, Bethesda, MD, USA

* To whom correspondence should be addressed. E-mail: chubn{at}ninds.nih.gov.

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 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 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 inter-episode 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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