Kroeger D, Tamburri A, Amzica F, Sik A. Activity-dependent layer-specific changes in the extracellular chloride concentration and chloride driving force in the rat hippocampus. J Neurophysiol 103: 1905-1914, 2010. First published February 3, 2010; doi: 10.1152/jn.00497.2009. The transmembrane distribution of chloride anions (Cl-) determines the direction of the Cl- flux through GAB(A) A receptors; this establishes whether GABA A receptor-mediated responses are hyperpolarizing or depolarizing in neurons. Thus an activity-dependent reduction in the efficacy of inhibitory responses can be the result of an activity-induced reduction of the Cl- driving force. Using Cl--sensitive electrodes, we measured the extracellular Cl- concentration ([Cl-](o)) in each layer of the hippocampus under control conditions and after stimulation. In the control condition, [Cl-](o) was lower within the CA1 region (112.9 +/- 1.3 mM; mean +/- SD) than the CA3/dentate gyrus areas (117.7 +/- 1.2 mM). Stimulation of CA3 pyramidal cells led to an increase in the [Cl-](o). The maximum values were observed in the stratum lacunosum-moleculare (253.4 +/- 51.1 mM) and in the hilus (261 +/- 43.7 mM), whereas in the granular cell layer, it reached only 159.5 +/- 41 mM. The stimulation-induced [Cl-](o) increase was followed by a period of decreasing [Cl-](o) that fell below the control values. The maximum undershoot (21.6 +/- 0.7 mM) was observed in the s. radiatum. Systemic application of the gap junction blocker carbenoxolone significantly decreased the stimulation-induced Cl- extrusion in the dentate gyrus but only slightly modified it in the CA1 area. Carbenoxolone also drastically reduced the Cl- clearance. The time constant of the Cl clearance was similar between layers (83.4 +/- 15.9 ms) but increased after carbenoxolone application (207.1 +/- 44.4ms). Stimulation-induced changes in the [Cl-](o) significantly decreased the Cl- driving force and resulted in large fluctuations between layers (Delta=9.4 mV). The lowest value was observed in the stratum radiatum of the CA1 and the hilar region (7.7 mV), whereas the highest value was calculated for the granule cell layer (16.3 mV). We suggest that a decrease of the extracellular space is mainly responsible for the rapid [Cl-](o) increase while the gap junction coupled astrocytic network plays a key role in the activity-dependent redistribution and clearance of Cl- across layers of the hippocampus.