Outwardly rectifying tonically active GABAA receptors in pyramidal cells modulate neuronal offset, not gain

Abstract

Hippocampal pyramidal cell excitability is regulated both by fast synaptic inhibition and by tonically active high-affinity extrasynaptic GABA(A) receptors. The impact of tonic inhibition on neuronal gain and offset, and thus on information processing, is unclear. Offset is altered by shunting inhibition, and the gain of a neuronal response to an excitatory input can be modified by changing the level of "background" synaptic noise. Therefore, tonic activation of GABA(A) receptors would be expected to modulate offset and, in addition, to alter gain through a shunting effect on synaptic noise. Here we show that tonically active GABA(A) receptors in CA1 pyramidal cells show marked outward rectification, while the peaks of IPSCs exhibit a linear current-voltage relationship. As a result, tonic GABA(A) receptor-mediated currents have a minimal effect upon subthreshold membrane potential variation due to synaptic noise, but predominantly affect neurons at spiking threshold. Consistent with this, tonic GABA(A) receptor-mediated currents in pyramidal cells exclusively affect offset and not gain. Modulation of tonically active GABA(A) receptors by fluctuations in extracellular GABA concentrations or neuromodulators acting on high-affinity receptors potentially provides a powerful mechanism to alter neuronal offset independently of neuronal gain.

Figure 1.

The tails, but not peaks, of evoked IPSCs (eIPSCs) in hippocampal pyramidal neurons show outward rectification. A, Family of eIPSCs traces at different holding potentials between −90 and +45 mV from one neuron demonstrating slower decay at positive potentials. B, Superimposed normalized I–V relationships of peak amplitudes (closed circles) and amplitudes of the eIPSC tails measured at two different time points (20 ms, gray circles; 40 ms, open circles) as indicated in A showing outward rectification of IPSC tails with greater rectification further from the peak (n = 7; data points are fitted either with linear or second-order polynomial function). Norm. ampl., Normalized amplitude. C, The RI of the GABA_AR-mediated current during the IPSC decay phase increases with time after stimulation (stim.). The bar on the graph shows the RI of the peak currents, indicating that they do not rectify (n = 7).

Figure 2.

Tonic GABA_AR-mediated currents in hippocampal pyramidal cells display marked outward rectification. A, GABA_A receptor block by picrotoxin produces a positive shift in holding current in pyramidal cells voltage clamped to −70 mV. Representative traces (left) and time course of normalized tonic current following application of picrotoxin (PTX; right; n = 7). B, I–V relationship of the tonically active GABA_ARs obtained from the difference in holding current during voltage ramps before and after perfusing picrotoxin (see Materials and Methods; n = 7; black trace represents averaged I–V curve, gray area is SEM). C, Similar rectification was obtained by measuring steady-state holding currents at different voltages using a step protocol (see Materials and Methods; n = 4).

Figure 3.

Rectification is time independent. A, Reversing the voltage ramp does not change the rectification of the tonically active GABA_ARs. Top, Example of a recording when the cell was slowly depolarized from −70 mV to +40 mV (ramp-up) and then hyperpolarized back to −70 mV (ramp-down). The I–V relationships of the up and down ramps are identical (bottom). B, Example of the time course of the picrotoxin-sensitive current obtained with a step command from −60 mV to +40 mV (the fast capacitance artifact was removed). The full trace is shown in the inset. Dashed vertical lines indicate time points at which the magnitude of tonic current was measured. PTX, Picrotoxin.

Figure 4.

Rectification of tonically active GABA_ARs is more pronounced with a physiological intracellular Cl⁻ concentration. A, I–V plot obtained using a step protocol with 8 mm intracellular Cl⁻ (n = 4). Data points are fitted with a Boltzmann function. Representative trace demonstrating no shift in holding current at −74 mV (top). B, The rectification due to unequal concentration of Cl⁻ inside and outside neurons cannot fully account for the rectification of GABA_ARs. The predicted I–V curve due to Goldman–Hodgkin–Katz rectification (red line) superimposed with normalized experimental curve (black) demonstrating significant additional rectification due to receptor properties. C, Slope conductance mediated by tonically active GABA_ARs calculated for the fitted I–V curve in A. PTX, Picrotoxin.

Figure 5.

Rectification persists when the extracellular GABA concentration is increased. A, B, Application of picrotoxin (PTX) in the presence of the GAT1 blocker 30 μm SKF-89976A (n = 8, A) or 20–30 μm GABA (n = 9, B) reveals a shift in the normalized holding current at −70 mV (left), and continued rectification (right). C, Summary plot of the effects of GABA application or blockade of GABA transport on the holding current at −70 mV. Increased GABA concentration in the slice leads to a similar increase in the tonic current following addition of GAT1 blocker or 20–30 μm GABA. D, Summary plot of RI (see Materials and Methods) with either the GAT1 blocker or 20–30 μm GABA. In the I–V plots, black traces represent mean values, while gray areas are SEM; *p < 0.05; **p < 0.01; ***p < 0.001, t test.

Figure 6.

The tonic GABA_AR-mediated conductances in hippocampal pyramidal cells modulate the offset, but not the gain, of the I–O function. A, Application of GABA (20–30 μm) in the presence of simulated synaptic noise (g_noise) shifts the I–O curve of a neuron to the right without affecting the slope of fitted sigmoid function. Subsequent application of picrotoxin (PTX, 100 μm) shifts the curve in the opposite direction beyond the control values without a change in slope. Shown are sample traces of the neuron's response (A₁) at three data points marked by the rectangle (A₂). Neither activation nor blockade of the tonic GABA_AR-mediated conductance produces significant change in the membrane potential (V_m) distribution (A₃; data from the same cell). B–E, Summary data for changes in the offset (B) of the I–O function demonstrating a significant effect produced by application of GABA (20–30 μm) or picrotoxin (100 μm) (n = 11 and 15, respectively). The change in the offset was measured as a change in the magnitude of input conductance that results in 50% probability of action potential generation. There were however no significant changes in the slope of spike probability curve (C; also measured at 50% probability of action potential firing) and normalized area under the curve (AUC) from spike probability = 0–1 (D). Changes in V_m RMS noise (E) with either GABA or picrotoxin application (n = 11 and 10, respectively). g_noise(2) and g_noise(3) (supplemental Fig. S3, available at www.jneurosci.org as supplemental material) were used in experiments. Open circles in B–E are values for individual cells; **p < 0.01; ***p < 0.001; paired t test.

Figure 7.

Rectifying tonic GABA_AR-mediated inhibition decreases its shunting effect on the background synaptic noise and thus favors control of offset over modulation of gain. A, Similar to the experimental paradigm, the modeled pyramidal cell receives two inputs simulating variable EPSPs (g_sEPSP) and stochastic synaptic noise (g_noise). Traces of the V_m at the cell soma resulting from the introduction of g_sEPSP and g_noise separately and combined (lower trace). B, An example of I–V curves for rectifying and nonrectifying inhibition simulated in the pyramidal cell model by introducing a tonic inhibitory conductance with a reversal potential E_i = −65 mV and g_max = 0.0001 S/cm². C, The conductances of rectifying (g_max = 15 nS, at +40 mV) and nonrectifying (g_const = 3 nS) tonic inhibition were adjusted to give the same offset of the I–O function in the absence of background synaptic noise (top). The effects of these “equivalent” rectifying and nonrectifying inhibitions on the I–O function were then compared in the presence of synaptic noise (bottom). D, The I–O curves are plotted without offset to compare the range of synaptic inputs that lead to the generation of action potentials. The curves are aligned to match the maximum spike probability point. The bar chart shows that rectification preserves the AUC values, in contrast to a constant tonic conductance, which decreases the AUC. The unitary excitatory conductance in the noise templates was 0.0021 μS for all simulations [corresponding to g_noise(3) in experiments]. E, The shunting effect of a constant tonic conductance on subthreshold noise is greater than that of rectifying inhibition. RMS is plotted against tonic conductance at +40 mV (g_max). Upper traces illustrate the effects of rectifying and nonrectifying tonic inhibition on RMS with g_max = 4 nS. F, The difference between the effects of rectifying and nonrectifying tonic inhibition on the variation of membrane potential is apparent even when their respective g_max values are matched to produce the same offset of I–O function in the absence of noise.