Chloride-sensitive MEQ fluorescence in chick embryo motoneurons following manipulations of chloride and during spontaneous network activity.
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 muM) 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 microM) 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.
Chub
Nikolai
N
Laboratory of Neural Control, NINDS/NIH, Rm. 3BC911, 35 Convent Dr., Bethesda, MD 20892-3700, USA. chubn@ninds.nih.gov
Mentis
George Z
GZ
O'donovan
Michael J
MJ
D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D001683:Biological Clocks / Q000502:physiology*; D002642:Chick Embryo; D002713:Chlorine / Q000378:metabolism*; D006912:Hydroxyquinolines; D015640:Ion Channel Gating / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods; D009046:Motor Neurons / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013116:Spinal Cord / Q000196:embryology / Q000502:physiology
D006912:Hydroxyquinolines; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002713:Chlorine