Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor

Abstract

The alpha5 subunit of the GABA(A) receptor is localized mainly to the hippocampus of the mammalian brain. The significance of this rather distinct localization and the function of alpha5-containing GABA(A) receptors has been explored by targeted disruption of the alpha5 gene in mice. The alpha5 -/- mice showed a significantly improved performance in a water maze model of spatial learning, whereas the performance in non-hippocampal-dependent learning and in anxiety tasks were unaltered in comparison with wild-type controls. In the CA1 region of hippocampal brain slices from alpha5 -/- mice, the amplitude of the IPSCs was decreased, and paired-pulse facilitation of field EPSP (fEPSP) amplitudes was enhanced. These data suggest that alpha5-containing GABA(A) receptors play a key role in cognitive processes by controlling a component of synaptic transmission in the CA1 region of the hippocampus.

Fig. 1.

Generation and validation of α5 −/− mice.a, b, Schematic representation of the WT α5 allele of the GABA_A receptor and the targeting vector, respectively.2, 3,4, Exons 2, 3, and 4; 2Δ,4Δ, partial deletion of exons 2 and 4; neo, neomycin resistance gene;TK, thymidine kinase gene; Nh,NheI restriction site; B,BamHI restriction site; P1,P2, PCR primers. c–g, Pharmacological and biochemical characterization of α5-deficient mice. c, d, Color-coded autoradiograms for [³H]L-655,708 binding to mouse brain sections reveals binding to the hippocampus in the WT (c) and absence of signal in the α5 −/− mice (d). Scale bar, 1 mm. e, Western blot shows the absence of a specific α5 subunit band at 55 kDa in membranes from α5 −/− hippocampus. f, Saturation experiment demonstrating the absence of high-affinity [³H]L-655,708 binding sites in α5 −/− mice (open circles) compared with WT mice (closed circles). g, Total number of [³H] BZ sites labeled by [³H]Ro15–1788 is reduced (−16%) in α5 −/− mice compared with WT controls.

Fig. 2.

Enhanced performance of α5 −/− mice in the matching-to-place version of the water maze test. Eighteen WT and 20 α5 −/− mice were used in this test. a, The difference in time taken between trial 1 and trial 2 (savings) to find the hidden platform over the 10 d testing period is shown. α5 −/− mice made significantly higher savings compared with WT mice.b, α5 −/− mice were significantly quicker (*) at finding the hidden platform for both trial 2 and trial 3.

Fig. 3.

Normal performance of the α5 −/− mice in the elevated plus maze. a, Data shown are mean (±SEM) values for percentage time spent in (left panel) and number of entries made to (right panel) different areas of the elevated plus-maze by WT and α5 −/− mice (n = 20–23). There were no significant differences between WT and α5 −/− mice on any of the measures. b, Data shown are mean (± SEM) values for percentage time spent in (left panel) and number of entries made to (right panel) different areas of the elevated plus-maze by 10.0 mg/kg CDP or vehicle-treated WT and α5 −/− mice (n = 13–20). CDP had a significant anxiolytic-like effect of the same magnitude in both WT and α5 −/− mice (*). This is indicated by the increase in the percentage of time spent in (p < 0.01) and percentage of entries made to (p < 0.01) the open arms of the plus-maze in comparison with vehicle-treated WT and α5 −/− mice.

Fig. 4.

Normal performance of the α5 −/− mice in the non-hippocampal-dependent two-way active avoidance. Data shown are mean (± SEM) avoid responses recorded on each of 12 d by untreated male (top) and female (bottom) wild-type (WT) and GABA_A receptor α5 knock-out (α5 −/−) mice (n = 7–10). There was no significant difference between WT and α5 −/− mice but a significant effect of sex (p < 0.05).

Fig. 5.

Synaptic strength in hippocampal brain slices of α5 −/− mice compared with its effect on paired-pulse facilitation in CA1 region and paired-pulse depression in the dentate gyrus. In all figures the filled circles represent data from age-matched WT mice, and open circles are from α5 −/− mice. No significant difference was found in the maximal fEPSP amplitude in the CA1 region. a, Forty-six slices from 16 WT controls and 48 slices from 16 α5 −/− mice or dentate gyrus.b, Forty-one slices from 16 WT controls and 42 slices from 16 α5 −/− mice. In the CA1 region the amplitude of fEPSPs was significantly enhanced during paired-pulse stimulus intervals of 100–300 msec. c, Filled circles, 46 slices from 16 WT controls; open circles, 48 slices from 16 α5 −/− mice. In comparison, paired-pulse depression of fEPSPs in the dentate gyrus remained unaffected. d, Filled circles, 41 slices from 16 WT controls; open circles, 42 slices from 16 α5 −/− mice.

Fig. 6.

Long-term potentiation in CA1 region of hippocampal brain slices from α5 −/− mice. LTP was induced by theta burst (4 stimuli at 100 Hz repeated 10 times every 200 msec). The amount of LTP was not different between WT and α5 −/− mice (filled circles, 32 slices from 8 WT mice;open circles, 38 slices from α5 −/− mice) at 1 hour after the theta burst (p = 0.18;F_(1,68) = 1.82).

Fig. 7.

Characteristics of IPSCs recorded from CA1 pyramidal neurons in hippocampal slices in vitroprepared from WT and α5 −/− mice. a, Examples of recordings of membrane current from CA1 neurons in WT and α5 −/− mice at a holding potential of −60 mV. The regular downward deflections are sIPSCs. b, Cumulative frequency distributions of the sIPSC amplitude and interevent interval in WT (black;n = 15 slices) and α5 −/− mice (red;n = 13 slices), showing a clear reduction in IPSC amplitude but no change in IPSC frequency in the α5 −/− mice. c, Representative sIPSCs recorded from CA1 pyramidal neurons in hippocampal brain slices from WT and α5 −/− mice. In the examples shown, the decay of the sIPSC recorded from the WT mouse was best fitted to a double-exponential function, whereas the decay of the sIPSC recorded from the α5 −/− mouse was best fitted to a monoexponential function (decay fits superimposed in red).d, Summary of the mean data of spontaneous and evoked IPSC mean peak amplitude and decay kinetics from WT and α5 −/− mice.