SUMMARY1. Intracellular Cl-activity (a',) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled C1--selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% 02, the resting membrane potential was -57-7 + 1'0 mV and a'I was 23-6 + 10 mm (n = 53). The value of a'1 was 2-6 times the activity expected for an equilibrium distribution and the difference between Em and Ec1 was 25 mV.2. Removal of external Cl-led to a reversible fall in a'c. Initial rates of decay and recovery of a', were 4-1 and 3-3 mm min-', respectively. During the recovery of a' following return to standard Ringer solution, most of the movement of Cl-occurred against the driving force for a passive distribution. Changes in ai, were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in a', when external Cl-was removed were too high to be accounted for by electrodiffusion.3. The intracellular accumulation of Cl-was dependent on the extracellular Clactivity (ao1). The relationship between at1 and aO I had a sigmoidal shape with a halfmaximal activation of about 50 mM-external Cl-.4. The steady-state a', depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl-after intracellular Cldepletion was abolished in the absence of either Na+ or K+ in the bathing solution.5. The reaccumulation of Cl-was inhibited by furosemide (0-5-1 x 10-3 M) or bumetanide (10-5 M). The decrease in a'i observed in Cl--free solutions was also inhibited by bumetanide.6. Cell volume changes were calculated from the observed changes in at,. Cells were estimated to shrink in Cl--free solutions to about 75 % their initial volume, at an initial rate of 6% min-'.* Address for correspondence and reprint requests.F. J. ALVAREZ-LEEFMANS AND OTHERS 7. The present results provide direct evidence for the active accumulation of Clin DRG neurones. The mechanism of Cl-transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+: K+: Cl-co-transport system mediates the active transport of Cl-across the cell membrane of DRG neurones.

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. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex.NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCCimmunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.