Heterogeneous chloride extrusion capacity in different regions of the spinal dorsal horn

GABA and glycine are the major inhibitory neurotransmitters in the CNS. GABAA and glycine receptors are mainly permeable to chloride (Cl-) ions. In adult neurons the K+-Cl- cotransporter isoform 2 (KCC2) maintains the intracellular Cl- concentration at a low level (<10mM). The Cl- reversal potential is therefore more negative than the resting membrane potential, making GABAA/glycine receptor-mediated currents hyperpolarizing. We have previously shown that a decrease in KCC2 activity in the spinal dorsal horn (DH) following peripheral nerve injury weakens Cl--mediated hyperpolarization, compromising local inhibitory control and causing pain hypersensitivity (Coull et al. Nature 2003, 2005). We have also shown that neurons with weaker Cl- extrusion capacity at certain developmental stages are more prone to a collapse in hyperpolarizing inhibition in conditions of repetitive activity (Cordero-Erausquin et al. J. Neurosci. 2005). OBJECTIVES. Here, we asked whether specific DH cell populations show differences in Cl- extrusion capacity in normal conditions and thus may be more or less sensitive to Cl- accumulation when challenged. MATERIALS AND METHODS. Parasagittal spinal cord slices were obtained from adult rats. Whole cell patch clamp recordings were obtained from lamina I and II neurons using low (9mM) or high (29mM) Cl- containing pipettes and reversal potential of GABA (EGABA) was measured in the presence of CNQX, APV and TTX. The expected EGABA, calculated with the Hodgkin-Katz-Goldman equation (taking into account HCO3-permeability), was -60mV and -37mV. Complementary experiments were performed using gramicidin-perforated patch clamp recordings to measure normal EGABA. Recorded neurons were filled with Lucifer yellow to allow laminar localization. RESULTS. Recordings from lamina I neurons in high Cl- condition yielded measured EGABA more negative than the expected value (-46±1mV, n=38). Values were distributed along a wide range (-35mV to -59mV), indicative of a substantial heterogeneity in Cl- extrusion capacity between cells. The KCC2 blocker furosemide (100µM) brought EGABA close to the expected value (-40±3mV; n=9, P<0.01), while bumetanide (10µM), a blocker of NKCC1 transporter, had no effect. In lamina II, EGABA measured using high Cl- pipettes was significantly more negative then in lamina I (-53±2mV, n=16; range -41mV to -69mV; P<0.01). In presence of furosemide lamina II EGABA was -42±2mV (n=4). This interlaminar difference was reduced when low Cl- pipettes were used (lamina I: -70±3mV, n=4; lamina II: -73±2mV, n=4; P=0.5). Preliminary data using gramicidine-perforated patch clamp show no significant differences in EGABA between the two cell populations (lamina I: -77±2mV, n=3; lamina II: -81±1mV, n=3; P=0.2). CONCLUSIONS. While both lamina I and II neurons appear to show comparable EGABA in normal conditions, imposing a Cl- load revealed a weaker Cl- extrusion capacity in lamina I neurons. These results show a higher level of KCC2 activity in lamina II suggesting that these neurons may be more resistant to Cl- accumulation when bombarded by repetitive activity. Funded by CIHR, FRSQ & Regione Piemonte