Alpha5GABAA receptor activity sets the threshold for long-term potentiation and constrains hippocampus-dependent memory

Abstract

Synaptic plasticity, which is the neuronal substrate for many forms of hippocampus-dependent learning, is attenuated by GABA type A receptor (GABA(A)R)-mediated inhibition. The prevailing notion is that a synaptic or phasic form of GABAergic inhibition regulates synaptic plasticity; however, little is known about the role of GABA(A)R subtypes that generate a tonic or persistent inhibitory conductance. We studied the regulation of synaptic plasticity by alpha5 subunit-containing GABA(A)Rs (alpha5GABA(A)Rs), which generate a tonic inhibitory conductance in CA1 pyramidal neurons using electrophysiological recordings of field and whole-cell potentials in hippocampal slices from both wild-type and null mutant mice for the alpha5 subunit of the GABA(A)R (Gabra5(-/-) mice). In addition, the strength of fear-associated memory was studied. The results showed that alpha5GABA(A)R activity raises the threshold for induction of long-term potentiation in a highly specific band of stimulation frequencies (10-20 Hz) through mechanisms that are predominantly independent of inhibitory synaptic transmission. The deletion or pharmacological inhibition of alpha5GABA(A)Rs caused no change in baseline membrane potential or input resistance but increased depolarization during 10 Hz stimulation. The encoding of hippocampus-dependent memory was regulated by alpha5GABA(A)Rs but only under specific conditions that generate moderate but not robust forms of fear-associated learning. Thus, under specific conditions, alpha5GABA(A)R activity predominates over synaptic inhibition in modifying the strength of both synaptic plasticity in vitro and certain forms of memory in vivo.

Figure 1.

α5GABA_ARs critically regulate the threshold for LTP within a narrow range of stimulus frequencies. Genetic deletion of the α5 subunit of the GABA_AR (Gabra5^−/−) lowered the threshold for synaptic plasticity for slices subjected to moderate-frequency stimulation but not low- or high-frequency stimulation. The effects of stimulation at 1 Hz (A), 100 Hz (B), and 10 Hz (C) on the slope of the fEPSP (percentage of baseline) are shown. Sample traces are shown above each figure for the times indicated by the numbers. Calibration: 0.5 mV, 10 ms. D, Persistent changes in synaptic strength were significant only for stimulation at 10 and 20 Hz. *p < 0.001.

Figure 2.

Normal synaptic transmission and excitability in α5 null mutant (Gabra5^−/−) slices. There were no differences between the genotypes for any of the following characteristics. A, The traces showed no difference between WT and Gabra5^−/− slices after stimulation at different intensities. B, Individual responses indicated that the input–output relationships were similar for WT and Gabra5^−/− slices, which suggests normal baseline presynaptic function and synaptic efficacy in the Gabra5^−/− mouse model. C, Sample traces show normal paired-pulse facilitation for each genotype. D, Grouped data show that facilitation of paired synaptic pulses (PSPs) with different interstimulus intervals (ISI) was the same for WT and Gabra5^−/− slices. E, Traces showing enhanced excitability of population spikes after application of bicuculline.

Figure 3.

Glutamate receptor currents and expression were not altered in α5 null mutant (Gabra5^−/−) neurons. A, AMPA (−80 mV) and NMDA (+40 mV) currents were recorded in WT and α5 null mutant (Gabra5^−/−) neurons. B, There was no difference in the peak NMDA to AMPA ratio in WT (n = 7) and Gabra5^−/− (n = 9) neurons for any of the stimulus intensities tested. C, AMPA currents, evoked at intensities ranging from 5 to 10 V, were the same in WT and Gabra5^−/− neurons. D, There was no difference in the peak amplitude of NMDA currents evoked at intensities ranging from 5 to 10 V in WT and Gabra5^−/− neurons. E, Left, Immunoblot assays for ionotropic glutamate receptor subtypes, including the NMDA receptor subunits NR1, NR2A, and NR2B and the AMPA receptor subunit GluR1, showed no difference in expression levels between WT and Gabra5^−/− mice. Right, The change in the level of receptor expression was determined from the ratio of total protein expressed in Gabra5^−/− tissue to total protein expressed in WT tissue. In the Gabra5^−/− tissue, the total amount of protein expressed did not change for glutamatergic receptor subtypes, and the ratio for total α5 was 0 because Gabra5^−/− tissue does not possess the α5 protein.

Figure 4.

Pharmacologic studies confirmed that α5GABA_ARs are critical for the induction of LTP after moderate-frequency but not high-frequency stimulation. Recording of fEPSPs under control conditions was followed by application of L-655,708 for 10 min before (A) or immediately after (B) 10 Hz stimulation. Application of L-655,708 before but not after 10 Hz stimulation enhanced the fEPSPs of WT slices, which suggests that α5GABA_ARs are critical for the induction of threshold LTP but not for maintenance of the response. C, We next measured overall involvement of GABA_ARs in the LTP of submaximal stimulation by blocking these receptors with the competitive antagonist bicuculline. Application of bicuculline to WT slices that had been stimulated at 10 Hz produced LTP that was indistinguishable from that observed in Gabra5^−/− slices but did not further enhance the LTP in Gabra5^−/−slices. D, Bicuculline further potentiated LTP in WT and Gabra5^−/− slices relative to drug-free conditions with 100 Hz stimulation, which suggests that GABA_ARs not containing the α5 subunit play an active role in LTP when the intensity of activation is increased. Black traces, Pretetanus baseline; blue and red traces, 60 min after tetanus in WT and Gabra5^−/−slices, respectively. Calibration: 0.5 mV, 10 ms.

Figure 5.

Blockade of synaptic GABA_ARs does not enhance plasticity with 10 Hz stimulation. A, Selectively blocking synaptically expressed GABA_ARs with SR-95531 (5 μm) did not enhance synaptic potentiation in WT (n = 8) or α5 null mutant (Gabra5^−/−) (n = 8) slices. B, A summary of changes in synaptic responses after 10 Hz stimulation. These data represent the average of the last 5 min of recording for each slice in each group. C, The IPSP (black) was blocked with SR-95531 before (light blue) and after (red) 10 Hz stimulation, which indicates that synaptic GABA_ARs could not account for the differences in plasticity at this frequency. D, Average data showing the raw amplitudes of the IPSP at baseline (without SR-95531), with SR-95531, and after 10 Hz stimulation for WT (n = 6) and Gabra5^−/− (n = 6) cells. *p < 0.01.

Figure 6.

α5GABA_ARs regulate membrane properties and the depolarizing envelope to set the threshold for long-term potentiation within the 10 Hz stimulation range. A, The membrane potential was significantly depolarized in α5 null mutant (Gabra5^−/−) and L-655,708-treated WT neurons during 10 Hz stimulation. The average membrane potential for the 10 min period after 10 Hz stimulation was significantly hyperpolarized from baseline values in WT. All recordings were conducted in the presence of SR-95531 and CGP 55845 to block synaptic GABA_A and GABA_B receptors, respectively. The membrane potential during and after 10 Hz stimulation were compared with baseline membrane potentials for the respective treatments. B, Sample traces show the membrane hyperpolarizing pulse for WT, Gabra5^−/−, and L-655,708-treated WT neurons, before and 30 min after 10 Hz stimulation. The average input resistance was significantly decreased in WT but not Gabra5^−/− or L-655,708-treated WT neurons. The individual changes in input resistance for each cell are shown in the “before” and “after” plots. Colored symbols represent the group means. 1, Baseline EPSP; 2, EPSP 30 min after 10 Hz stimulation. C, Sample traces show that, during 10 Hz stimulation, the EPSPs were significantly smaller, for the duration of the simulation period, in WT neurons than in Gabra5^−/− and L-655,708-treated WT neurons. Insets show that the EPSPs were larger for Gabra5^−/− and L-655,708-treated WT neurons near the beginning and at the end of the stimulation period. D, Pooled data show that the average depolarizing envelope was significantly larger in Gabra5^−/− neurons and L-655,708-treated WT neurons than in WT control neurons. E, Examples of EPSP responses for single WT (blue), Gabra5^−/− (red), and L-655,708-treated WT (gray) neurons. 1, Baseline EPSP; 2, EPSP 30 min after 10 Hz stimulation. Calibration: 4 mV, 10 ms. F, Summary of the EPSP changes after 10 Hz stimulation for all groups. *p < 0.05.

Figure 7.

α5GABA_ARs physiologically regulate the acquisition of weak hippocampus-dependent associative fear memory tasks. A, A schematic representation showing the timing for all three fear-conditioning protocols. In all protocols, a baseline activity period of 3 min preceded the conditioning procedure. Three 2 s, 0.5 mA footshocks, separated by 60 s intervals, were used for contextual fear conditioning. Three tone–shock pairings (20 s, 70 dB tone paired with a 2 s, 0.5 mA footshock), separated by 60 s intervals, were used for auditory cued fear conditioning. The procedure for trace fear conditioning was similar to that for cued fear conditioning (three tone–shock pairings, separated by 240 s intervals), except that an empty trace interval of 20 s was interposed between the tone and the footshock. B, There was no difference between WT (n = 9) and α5 null mutant (Gabra5^−/−) (n = 11) mice for contextual fear conditioning, which forms strong hippocampus-dependent memories; furthermore, L-655,708 had no effect on the freezing response (WT, n = 10; Gabra5^−/−, n = 11). C, The WT (n = 9) and Gabra5^−/− (n = 11) mice had similar scores during the amygdala-dependent cued fear-conditioning task. D, The performance of Gabra5^−/− (n = 12) mice was enhanced in trace fear conditioning (a weak associative task), relative to the effect in naive (n = 12) and vehicle-treated (n = 13) WT mice; in addition, inhibiting α5GABA_ARs with L-655,708 improved the performance of WT mice (n = 13) to the level observed in Gabra5^−/− mice. E, The performance of WT mice (n = 9) was not enhanced with L-655,708 injections immediately after training in the trace fear-conditioning protocol, relative to Gabra5^−/− mice (n = 8). *p < 0.05, significantly different from the control group.