Role of bicarbonate and chloride in GABA- and glycine-induced depolarization and [Ca2+]i rise in fetal rat motoneurons in situ

Abstract

Ca(2+) imaging and (perforated) patch recording were used to analyze the mechanism of GABA- and glycine-induced depolarizations in lumbar motoneurons of spinal cord slices from fetal rats. In fura-2 ester-loaded cells, the agonist-induced depolarizations increased [Ca(2+)](i) by up to 100 nm. The GABA- and glycine-evoked [Ca(2+)](i) transients were suppressed by bicuculline and strychnine, respectively. Their magnitude decreased by approximately 50% between embryonic days 15.5 and 19.5. The [Ca(2+)](i) increases were abolished by Ca(2+)-free superfusate and attenuated by approximately 65% by nifedipine, showing that the responses were mediated by voltage-activated Ca(2+) channels. The [Ca(2+)](i) rises were potentiated by >300% immediately after removal of Cl(-) from the superfusate but recovered to values of 50-200% of control during repeated agonist administration in Cl(-)-free saline. Bumetanide gradually suppressed the [Ca(2+)](i) increases by >75%. Subsequent removal of Cl(-) reconstituted the responses and increased, upon repeated agonist application, the peak [Ca(2+)](i) rises to values above control. Removal of HCO(3)(-) from the Cl(-)-free (bumetanide-containing) superfusate reversibly abolished both the agonist-induced [Ca(2+)](i) rises and depolarizations that were reestablished by formate anions. In Cl(-)-containing superfusate, removal of HCO(3)(-) decreased both the peak and duration of the agonist-evoked membrane depolarization and [Ca(2+)](i) response. Our findings show that HCO(3)(-) efflux has a major contribution to depolarizations mediated by GABA(A) and glycine receptor-coupled anion channels in prenatal neurons. We hypothesize that the HCO(3)(-)-dependent depolarizing component, which is likely to produce an intracellular acidosis, might play an important role during the early postnatal period when the Cl(-)-dependent component gradually shifts to hyperpolarization.

Fig. 1.

Increases of [Ca²⁺]_i in lumbar motoneurons of rat embryonic spinal cord slices. A, Tetanic antidromic stimulation (TS; 50 V, 50 Hz, 2 sec) of the ipsilateral ventral nerve rootlet evokes a noticeable rise of [Ca²⁺]_i that is abolished by 0.5 μm TTX. In contrast, the more moderate [Ca²⁺]_i increase in response to bath application (20 sec) of 200 μm GABA is not affected by TTX. Traces correspond to somatic measurements in the regions of interest indicated in the micrograph. B, In a different spinal cord slice, TTX has no effect on the [Ca²⁺]_i increase upon bath application (20 sec) of glycine (1 mm). C, Statistical analysis of the effects of TTX on [Ca²⁺]_i rises caused by TS, GABA, and glycine. Numbers indicate the population of measured cells. Means ± SEM.

Fig. 2.

Age-dependence of agonist-induced [Ca²⁺]_i increases. A, In a spinal cord slice from an E15.5 rat, bath-applied (20 sec) GABA (200 μm), glycine (1 mm), and glutamate (100 μm) elevate motoneuronal [Ca²⁺]_i by between 25 and 100 nm (top trace). Middle traceexemplifies that the response to glutamate is greatly potentiated in a slice from an E17.5 rat, whereas the [Ca²⁺]_i increases caused by GABA and glycine are smaller than at E15.5. Bottom traceillustrates that the GABA- and the glycine-induced [Ca²⁺]_i rises are even less pronounced in a preparation from an E19.5 rat. Also, the response to glutamate is smaller than at E17.5. B, Statistical analysis of the agonist-evoked [Ca²⁺]_i responses. Note the different scale for the glutamate effects.

Fig. 3.

Dose relationship of agonist-evoked [Ca²⁺]_i increases. A, Original recording of [Ca²⁺]_i rises caused by bath application (20 sec) of GABA. B, Statistical analysis reveals that the response to GABA saturates at concentrations >200 μm. C, Original recording of [Ca²⁺]_i rises in response to bath application (20 sec) of glycine. D, Statistical analysis reveals that the response to glycine saturates at concentrations >250 μm.

Fig. 4.

Pharmacology of agonist-induced [Ca²⁺]_i increases. A, Bath-applied (20 sec) muscimol (10 μm) mimics the response to GABA (200 μm). B, Bicuculline (100 μm) reversibly abolishes the GABA-evoked [Ca²⁺]_i rise. C, The response to glycine (1 mm) is suppressed by strychnine (10 μm). D, Bicuculline does not evoke a major suppression of the [Ca²⁺]_i increase caused by glycine, whereas strychnine does not influence the response to GABA. E, Statistical analysis of the percentage antagonist-induced reduction of the GABA and glycine responses.F, Statistical analysis of the response to baclofen and muscimol, represented as the percentage of the peak [Ca²⁺]_i rise elicited by GABA.

Fig. 5.

Mediation of anion channel-induced [Ca²⁺]_i increases by voltage-activated Ca²⁺ channels. A, The [Ca²⁺]_i transient caused by bath-applied GABA (200 μm) is suppressed in nominally Ca²⁺-free superfusate. B, Ca²⁺-free saline also abolishes the [Ca²⁺]_i rise in response to glycine (1 mm). C, The Ca²⁺ channel antagonist nifedipine (50 μm) reduces the glycine-evoked [Ca²⁺]_i response. D, Statistical analysis of the blocking effects of Ca²⁺-free saline and nifedipine on agonist-induced [Ca²⁺]_i rises.

Fig. 6.

Anion-dependence of agonist-evoked [Ca²⁺]_i increases. A,B, Shortly after introduction of a nominally Cl⁻-free superfusate, the [Ca²⁺]_i rise in response to bath-applied GABA (200 μm; A) or glycine (1 mm; B) is potentiated several-fold.C, HCO₃⁻-free saline abolishes the [Ca²⁺]_i transient evoked by GABA (200 μm) after its potentiation by Cl⁻-free superfusate. Subsequent addition of formate anions (10 mm) fully restores the response.C, Statistical analysis of the initial potentiation (left columns) and the steady-state (right columns) effect of Cl⁻-free superfusate on the agonist-induced [Ca²⁺]_i increases represented as percentage of control. Note the differences in the scale of the ordinates.

Fig. 7.

Attenuation of anion channel-mediated [Ca²⁺]_i rises by HCO₃⁻-free superfusate.A, CO₂/HCO₃⁻-free superfusate reduces both the peak amplitude and duration of [Ca²⁺]_i rises elicited by glycine (1 mm) or GABA (200 μm). B, In a different slice, omission of CO₂/HCO₃⁻ does not reduce the response to glutamate (100 μm).C, Statistical analysis of the effects of CO₂/HCO₃⁻-free saline on the peak of the [Ca²⁺]_i rise represented as percentage of the control agonist response.

Fig. 8.

Role of an inwardly directed Cl⁻ pump in anion channel-mediated [Ca²⁺]_i increases. A, The Na⁺/K⁺/2Cl⁻-cotransport blocker bumetanide (100 μm) attenuates the [Ca²⁺]_i rise caused by glycine (1 mm). B, In a different preparation, bumetanide leads to a more moderate depression of the [Ca²⁺]_i transient caused by GABA (200 μm). The remaining component is almost abolished by CO₂/HCO₃⁻-free superfusate. C, After bumetanide-related suppression of the GABA-induced [Ca²⁺]_i rise, Cl⁻-free solution exerts a potentiating effect that is attenuated by CO₂/HCO₃⁻-free saline. Note that the bumetanide-dependent attenuation of the [Ca²⁺]_i rise was not attributable to rundown because the response to the agonists remained stable during repeated application for time periods >1 hr (compare with Fig.3).

Fig. 9.

Perforated-patch recording of anion channel-mediated depolarizations. A, HCO₃⁻-free saline attenuates and shortens the depolarization in response to bath-applied GABA (200 μm). B, Statistical analysis of the depolarization induced by GABA and of the attenuating effect of CO₂/HCO₃⁻-free saline. C, Cl⁻-free superfusate increases the peak of the GABA depolarization. After introduction of HCO₃⁻-free solution, the GABA depolarization is abolished but reappears upon addition of formate anions (10 mm).

Fig. 10.

Whole-cell recording of the HCO₃⁻-dependent component of the response to GABA. After rupture of a gramicidin perforated patch (low Cl⁻ patch solution), GABA (200 μm) evokes a hyperpolarization and decrease of input resistance. The response shifts to a depolarization in Cl⁻-free superfusate and is reversibly blocked by removal of HCO₃⁻.