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

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

FERRINI, Francesco Maria;
2009

Abstract

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
3rd Annual Canadian Neuroscience Meeting
Vancouver (Canada)
24-27 Maggio, 2009
Abstracts of 3rd Canadian Neuroscience Meeting
CD-ROM
CD-ROM
midollo spinale; KCC2; sinapsi GABAergiche; nocicezione
Ferrini F; De Konicnk Y
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/81866
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