Regulation of GABAergic inputs to CA1 pyramidal neurons by nicotinic receptors and kynurenic acid

Abstract

Impaired α7 nicotinic acetylcholine receptor (nAChR) function and GABAergic transmission in the hippocampus and elevated brain levels of kynurenic acid (KYNA), an astrocyte-derived metabolite of the kynurenine pathway, are key features of schizophrenia. KYNA acts as a noncompetitive antagonist with respect to agonists at both α7 nAChRs and N-methyl-D-aspartate receptors. Here, we tested the hypothesis that in hippocampal slices tonically active α7 nAChRs control GABAergic transmission to CA1 pyramidal neurons and are sensitive to inhibition by rising levels of KYNA. The α7 nAChR-selective antagonist α-bungarotoxin (α-BGT; 100 nM) and methyllycaconitine (MLA; 10 nM), an antagonist at α7 and other nAChRs, reduced by 51.3 ± 1.3 and 65.2 ± 1.5%, respectively, the frequency of GABAergic postsynaptic currents (PSCs) recorded from CA1 pyramidal neurons. MLA had no effect on miniature GABAergic PSCs. Thus, GABAergic synaptic activity in CA1 pyramidal neurons is maintained, in part, by tonically active α7 nAChRs located on the preterminal region of axons and/or the somatodendritic region of interneurons that synapse onto the neurons under study. L-Kynurenine (20 or 200 μM) or KYNA (20-200 μM) suppressed concentration-dependently the frequency of GABAergic PSCs; the inhibitory effect of 20 μM L-kynurenine had an onset time of approximately 35 min and could not be detected in the presence of 100 nM α-BGT. These results suggest that KYNA levels generated from 20 μM kynurenine inhibit tonically active α7 nAChR-dependent GABAergic transmission to the pyramidal neurons. Disruption of nAChR-dependent GABAergic transmission by mildly elevated levels of KYNA can be an important determinant of the cognitive deficits presented by patients with schizophrenia.

Fig. 1.

Spontaneous GABAergic PSCs from CA1 pyramidal neurons. A, sample recordings of spontaneous GABAergic PSCs at 0 mV obtained from CA1 pyramidal neurons of 30-day-old rats under the control condition (top trace). The second and third traces show spontaneous PSCs at an expanded time scale under the control condition. Bottom trace shows recordings at 0 mV 15 min after superfusion of the slice with ACSF containing the GABA_A receptor antagonist bicuculline (10 μM). B, representative recordings of PSCs from another neuron at 0 mV before (top trace) and during superfusion of slices with ACSF containing glutamate receptor antagonists APV (50 μM) and CNQX (10 μM) (bottom trace). Neurons had been superfused with the glutamate receptor antagonists for 15 min before beginning analysis. C, quantification of the effects of bicuculline and APV + CNQX on the spontaneous PSCs recorded from CA1 pyramidal neurons at 0 mV. Graph and error bars represent mean and S.E.M., respectively, of data obtained from five neurons from four rats in bicuculline and six neurons from three rats in CNQX + APV.

Fig. 2.

Effect of MLA on frequency of GABAergic PSCs. A, cumulative probability plots of interevent intervals and peak amplitude (inset) of PSCs recorded from control and MLA-incubated slices. Plots represent data from five neurons from four rats for control and eight neurons from four rats for MLA incubation. MLA caused a significant rightward displacement of the cumulative distribution of interevent intervals (p < 0.01 according to K-S test). B, mean frequency of GABAergic PSCs recorded 1) under control conditions and 2) in the continuous presence of MLA after 2-h incubation with MLA. Graph and error bars represent mean and S.E.M., respectively, of data obtained from 10 neurons from five rats in control and eight neurons from five rats in MLA. ***, p < 0.001 compared with control according to unpaired t test. C, mean frequency of GABAergic PSCs recorded under control condition followed by 15-min superfusion of the slices with MLA. Graph and error bars represent mean and S.E.M., respectively, of data obtained from seven neurons from seven rats. ***, p < 0.001 compared with control according to paired t test.

Fig. 3.

Effect of α-BGT on frequency of GABAergic PSCs. A, cumulative probability of interevent intervals of PSCs recorded under control conditions and after 1-h incubation in ACSF containing 100 nM α-BGT. Plots represent data from four neurons from four rats in control and eight neurons from four rats in α-BGT. The cumulative distribution of interevent intervals obtained in the presence of α-BGT was significantly displaced to the right in comparison with control (p < 0.01 according to K-S test). B, quantification of the effects of α-BGT, CNQX + APV, and CNQX + APV + α-BGT. Results obtained from control slices were compared with those obtained from slices after 1-h incubation with CNQX + APV or 1-h incubation with CNQX + APV + α-BGT. Graph and error bars represent mean and S.E.M., respectively, of data obtained from five neurons from five rats in the control condition, eight neurons from five rats in α-BGT incubation, five neurons from three rats in CNQX + APV incubation, and six neurons from three rats in CNQX + APV + α-BGT. **, p < 0.01 compared with control according to unpaired t test. ##, p < 0.01 compared with CNQX + APV by unpaired t test. C, frequency of PSCs recorded in the continuous presence of α-BGT (100 nM) was compared with that recorded in the continuous presence of α-BGT (100 nM) + MLA (10 nM). In these experiments, all slices were incubated for 1 h in α-BGT (100 nM)-containing ACSF and subsequently superfused with ACSF containing only α-BGT (100 nM) or both α-BGT (100 nM) and MLA (10 nM). Graph and error bars represent mean and S.E.M., respectively, of data obtained from seven neurons from four rats in α-BGT and seven neurons from four rats in α-BGT + MLA. *, p < 0.01 according to paired t test.

Fig. 4.

Effect of TTX on the frequency of GABAergic PSCs. A, cumulative distribution of interevent intervals of PSCs recorded under control conditions, in the continuous presence of 200 nM TTX, or in the continuous presence of 200 nM TTX + 10 nM MLA. In comparison with control, the cumulative distribution of interevent intervals obtained in the presence of TTX or TTX + MLA was displaced to the right (p < 0.001 according to K-S test). B, mean frequency of PSCs recorded under the same experimental conditions as in A. Graph and error bars represent mean and S.E.M., respectively, of data obtained from five neurons from five rats in control, 10 neurons from six rats in TTX, and six neurons from four rats in TTX + MLA. ***, p < 0.001 compared with control according to one-way ANOVA followed by Dunnett post hoc test.

Fig. 5.

Concentration-dependent effect of kynurenine (Kyn) on the frequency of spontaneous PSCs. A, cumulative probability plot of interevent intervals of PSCs recorded under control conditions or in the continuous presence of kynurenine (2–200 μM) after 2- to 5-h incubation in ACSF containing the corresponding concentration of kynurenine. The plots were obtained from data in 11 neurons from 11 rats in control, seven neurons from four rats in 2 μM kynurenine, 10 neurons from six rats in 20 μM kynurenine, and six neurons from four rats in 200 μM kynurenine. Cumulative distributions of interevent intervals recorded from neurons in the presence of 20 and 200 μM kynurenine were significantly displaced to the right in comparison with control (p < 0.05 and 0.01, respectively, according to K-S test) compared with control. B, mean frequency of PSCs recorded 1) under control conditions, 2) in the continuous presence of 2, 20, or 200 μM kynurenine after 2- to 5-h incubation with the corresponding concentration of kynurenine, or 3) during 15-min perfusion with 200 μM kynurenine. Compared with control, kynurenine reduced significantly the mean frequency of PSCs: *, p < 0.05; **, p < 0.01 according to one-way ANOVA followed by Dunnett post hoc test. The magnitude of the effect of 200 μM kynurenine was larger than that of 20 μM kynurenine (#, p < 0.05 according to one-way ANOVA followed by Tukey post hoc test). Graph and error bars represent mean and S.E.M., respectively, of data obtained from same number of neurons and rats as in A. Data from five neurons from three rats were used in the 200 μM kynurenine bath application. C, graph shows time-dependent percentage reduction in PSC frequency by continuous superfusion of ACSF containing kynurenine (20 μM). The maximum inhibition obtained during an incubation protocol (data from B) is provided in the last column for comparison. The onset time for the effect kynurenine was ∼35 min. *, p < 0.05; **, p < 0.01 according to one-way ANOVA followed by Dunnett post hoc test.

Fig. 6.

Concentration-dependent effect of exogenously applied KYNA on the frequency of spontaneous PSCs. A, mean frequency of PSCs recorded under control condition or in the continuous presence of KYNA (0.1–200 μM) after 2- to 5-h incubation in ACSF containing the corresponding concentration of KYNA. Graph and error bars represent mean and S.E.M., respectively, of data obtained from 17 neurons from 17 rats in control, four neurons from three rats in 100 nM KYNA, six neurons from three rats in 1 μM KYNA, eight neurons from five rats in 20 μM KYNA, six neurons from four rats in 50 μM KYNA, five neurons from three rats in 100 μM KYNA, and six neurons from four rats in 200 μM KYNA. *, p < 0.05; **, p < 0.01 compared with control according to one-way ANOVA followed by Dunnett post hoc test. B, mean frequency of PSCs recorded in the continuous presence of TTX (200 nM) or in the continuous presence of TTX (200 nM) plus KYNA (20 μM). Graph and error bars represent mean and S.E.M., respectively, of data obtained from 12 neurons from eight rats in TTX and five neurons from two rats in TTX + KYNA. C, graph showing time-dependent percentage reduction in PSC frequency by continuous superfusion of ACSF containing KYNA (200 μM). In this set of experiments, ACSF contained 10 μM CNQX + 50 μM APV during and 10 min before application of KYNA. The onset time for the effect KYNA was approximately 15 min. **, p < 0.01 according to one-way ANOVA followed by Dunnett post hoc test.

Fig. 7.

Effect of the admixture of kynurenine and α-BGT on the frequency of PSCs. A, mean frequency of PSCs recorded 1) after 1-h incubation with α-BGT (100 nM), 2) after 1-h incubation with ACSF containing α-BGT (100 nM) followed by 2- to 5-h incubation with α-BGT (100 nM) plus kynurenine (20 μM), or 3) after 1-h incubation with ACSF containing α-BGT (100 nM) followed by 2- to 5-h incubation with α-BGT (100 nM) + kynurenine (200 μM). In each experimental group, the ACSF used to superfuse the slices was the same as that used during the incubation time. Graph and error bars represent mean and S.E.M., respectively, of data obtained from 10 neurons from six rats in α-BGT, six neurons from four rats in α-BGT + 20 μM kynurenine, and six neurons from four rats in α-BGT + 200 μM kynurenine. The effect of admixture of α-BGT (100 nM) + kynurenine (200 μM) is significantly higher than that of α-BGT alone (**, p < 0.01 according to one-way ANOVA followed by Tukey post hoc test). B, graph shows the percentage reduction of PSC frequency in the continuous presence of α-BGT (100 nM), kynurenine (20 μM), α-BGT (100 nM) + kynurenine (20 μM), kynurenine (200 μM), or α-BGT (100 nM) + kynurenine (200 μM). The magnitude of effect of admixture of α-BGT (100 nM) + kynurenine (20 μM) was significantly larger than kynurenine (20 μM) alone and not different from that of α-BGT (100 nM). The effect of α-BGT (100 nM) + kynurenine (200 μM) was significantly larger than that of kynurenine (200 μM) or α-BGT (100 nM) alone. ** and ##, p < 0.01 according to one-way ANOVA followed by Tukey post hoc test.

Fig. 8.

Combined effect of kynurenine and MLA on the frequency of PSCs. A, cumulative probability plots of interevent intervals of PSCs recorded under the control condition and after 2- to 5-h incubation in ACSF containing different agents. In each experimental group, the ACSF used to superfuse the slices was the same as that used during the incubation time. Compared with control, cumulative distributions of interevent intervals were displaced to the right by 10 nM MLA (p < 0.001, according to K-S test), 200 μM kynurenine (p < 0.01, according to K-S test), and 200 μM kynurenine + 10 nM MLA (p < 0.001, according to K-S test). Cumulative probability plots of data were obtained from 19 neurons from 19 rats in control, eight neurons from four rats in MLA, six neurons from four rats in 200 μM kynurenine, and eight neurons from four rats in 200 μM kynurenine + 10 nM MLA. B, mean frequency of PSCs recorded under the same experimental conditions as in A. Graph and error bars represent mean and S.E.M., respectively, of data obtained from the same number of neurons as in A. In the presence of 200 μM kynurenine, 200 μM kynurenine + 10 nM MLA and 10 nM MLA, the mean frequency of PSCs was significantly lower than that of control (***, p < 0.001 according to one-way ANOVA followed by Dunnet post hoc test). Mean frequency of PSCs recorded in the presence of 200 μM kynurenine + 10 nM MLA was significantly lower than that recorded in the presence of kynurenine (200 μM) or MLA (10 nM) alone (#, p < 0.05 according to one-way ANOVA followed by Tukey post hoc test).