Contribution of subsaturating GABA concentrations to IPSCs in cultured hippocampal neurons

Abstract

The time course of EPSCs and IPSCs is at least partly determined by the concentration profile of neurotransmitter acting on postsynaptic receptors. Several recent reports have suggested that the peak synaptic cleft concentration of the inhibitory neurotransmitter GABA likely reaches at least 500 microM, a level that saturates the GABAA receptor. In the course of investigating the experimental anticonvulsant 3,3-diethyl-2-pyrrolidinone (diethyl-lactam), we have observed an important contribution to IPSC decay by subsaturating concentrations of GABA. Diethyl-lactam augments currents elicited by the exogenous application of subsaturating concentrations of GABA in voltage-clamped, cultured hippocampal neurons and significantly prolongs the decay of autaptic IPSCs and miniature IPSCs in our cultures. In addition, diethyl-lactam potentiates currents in excised outside-out membrane patches elicited by the prolonged application of low concentrations of GABA. However, when patches are exposed to 1-2 msec pulses of 1 mM GABA, diethyl-lactam does not alter current decay. Tiagabine, which blocks GABA reuptake, does not prolong IPSCs, so it is unlikely that uptake inhibition accounts for the enhancement of IPSCs. EPSCs and miniature IPSC frequency are unaffected by diethyl-lactam, again consistent with a postsynaptic site of action. We propose that during an IPSC, a substantial number of postsynaptic receptors must be exposed to subsaturating concentrations of GABA. A simplified model of GABAA receptor kinetics can account for the effects of diethyl-lactam on exogenous GABA and IPSCs if diethyl-lactam has its main effect on the monoliganded states of the GABAA receptor.

Fig. 1.

Chemical structures of α-substituted γ-butyrolactone and diethyl-lactam.

Fig. 2.

Diethyl-lactam modulation of whole-cell GABA_A currents. A, Hippocampal neurons were voltage clamped at −60 mV and exposed to exogenous application of 3 μm GABA ± 1 mm diethyl-lactam. Sample currents are shown. Calibration: 200 pA, 500 msec. B, Diethyl-lactam exhibited a concentration-dependent potentiation of peak currents with an apparent EC₅₀ of 2.2 mm. Each point represents the mean ± SE of at least five cells.C, Diethyl-lactam altered the GABA concentration–response curve by augmenting currents elicited by low but not saturating concentrations of GABA. Each point represents the mean ± SE of at least five cells. All currents are normalized to those elicited by 300 μm GABA without drug. For GABA alone, EC₅₀ and Hill coefficient were 15.3 and 1.2 μm, respectively. In the presence of 10 mmdiethyl-lactam, the EC₅₀ was 13.8 μm and the Hill coefficient was 0.9 μm.

Fig. 3.

Diethyl-lactam effects on synaptic transmission.A, Sample IPSC in the presence of control, 1 and 3 mm diethyl-lactam solutions. B, Sample EPSC in control and 1 mm diethyl-lactam solutions.C, Current-clamped microisland neurons adjusted to potentials of −60 mV, and then stimulated with 5 msec depolarizing current steps exhibited action potentials and resulting autaptic postsynaptic potentials. Action potentials were not altered by diethyl-lactam. Calibration: 5 msec, 20 mV.

Fig. 4.

Diethyl-lactam modulation of mIPSC duration.A, Spontaneous currents were recorded in five cells using solutions containing TTX, NBQX, and APV. Shown are sample mIPSCs recorded under control conditions (top) and in the presence of 1 mm diethyl-lactam (bottom). Calibration bar, 50 msec and 200 pA. B, The half-decay time (t_½) for each detectable independent miniature event was acquired and displayed in a cumulative probability plot. A representative plot from one cell is displayed, showing 1290 miniature events recorded under control conditions and 683 recorded in the presence of 1 mm diethyl-lactam. In every cell studied, 1 mm diethyl-lactam elicited a statistically significant increase in half-decay time (p< 0.05; Kolmogorov–Smirnov test). The currents displayed inA and summarized in B were recorded from different cells.

Fig. 5.

Rapid application of saturating GABA to excised patches. A, Sample currents in an excised patch elicited by brief (1.6 msec) applications of 1 mm GABA ± 1 mm diethyl-lactam. In the GABA + diethyl-lactam exposures, diethyl-lactam was present in the ECF barrel of the theta tubing, so the patches were exposed to the compound before the brief application of GABA + diethyl-lactam. The drug had no effect on the current decay. B, Patch-current decay was prolonged by 300 μm phenobarbital. C, Ratio oft_½,drug tot_½,control plotted versus t_½,control. Diethyl-lactam did not prolong t_½even in those patches that had fast controlt_½ values, therefore the phenobarbital effect cannot be explained by selection for patches with rapid current decay.

Fig. 6.

Diethyl-lactam effects on subsaturating GABA currents in patches. A, Sample current from an excised patch elicited by the prolonged application of 3 μmGABA ± 1 mm diethyl-lactam. Calibration: 20 pA, 200 msec. B, Sample current from another excised patch elicited by the prolonged application of 3 μm GABA ± 10 mm diethyl-lactam. Calibration: 50 pA, 200 msec. Patch excision does not render GABA_A receptors insensitive to modulation.

Fig. 7.

Simulation of diethyl-lactam effects on GABA_A receptors. A, Previously published kinetic scheme for GABA_A receptor activation, deactivation, and desensitization (Jones and Westbrook, 1995). Values for the control rate constants are as follows: k_F, 3 μm⁻¹; k_B, 150; d₁, 13;r₁, 0.13;d₂, 750;r₂, 15;C₁, 1111;O₁, 200;C₂, 142; andO₂, 2500, all sec⁻¹. In our model, diethyl-lactam increasesO₁ from 200 to 610 sec⁻¹. B, Concentration–response curves calculated for GABA and GABA + diethyl-lactam. The peak currents induced by a simulated step of GABA (400 msec) were calculated at concentrations between 1 and 300 μm GABA. The points were then fit by a logistic equation to give the two continuous lines. The control EC₅₀ is 18.4, and the Hill coefficient is 1.1 μm; diethyl-lactam reduces the EC₅₀ to 11.0 and the Hill coefficient to 1.0 μm (compare with Fig.2C). C, IPSC simulated by a GABA transient that decays as the sum of two exponentials (top trace, amplitudes 750 and 10 μm; time constants, 800 μsec and 133 msec, respectively). The gap in the display of the GABA transient (top trace) is necessary because the difference in the amplitudes of the two summed components is so large. The simulated addition of diethyl-lactam prolongs the decay of the IPSC. D, Patch currents simulated by a step application of GABA (1 mm for 1 msec, top trace). The presence of diethyl-lactam had virtually no effect on current deactivation because the two separate current traces in the figure completely superimpose.