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 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.

Dual personality of GABA/glycine-mediated depolarizations in immature spinal cord

“…In addition, there is increasing evidence that different subcellular compartments of individual neurons can express two distinct types of cation-chloride cotransporters so that a single neurotransmitter depolarizes or hyperpolarizes the different compartments (27,50). Consistent with this observation, changes in [Cl Ϫ ] i during spontaneous motor episodes are larger in MN dendrites than at the soma level in the chicken embryo (49). The present results show that all these variations may have profound consequences on the excitability of the soma and dendrites and the integration of excitatory inputs.…”

“…An important point in the two previously proposed mechanisms is the characteristic slow decay of the dIPSPs in immature MNs (45), which offers a large time window for the facilitation of excitatory events. (44,49). Similarly, a down-regulation of KCC2 in pathological conditions can cause a Ϸ20-mV positive shift of E Cl (26).…”

GABA A transmission is a critical step in the process of triggering homeostatic increases in quantal amplitude

“…In the present work, we block GABAergic or glutamatergic neurotransmission in the developing spinal cord in vivo while maintaining significant levels of SNA caused by the fast compensatory nature of the highly excitable developing cord (20,21). We have found a particular importance of GABA A transmission in triggering compensatory changes in quantal amplitude.…”

Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors

“…These compounds have low biological toxicity, a rapid response to changes in Cl and a relatively good sensitivity to Cl. However, they are prone to a time-dependent decay in fluorescence owing to the gradual leakage of dye from the labelled cells or bleaching (Krapf et al 1988;Biwersi & Verkman 1991;Inglefield & Schwartz-Bloom 1997;Nakamura et al 1997;Chub et al 2006;Painter & Wang 2006).…”