Age dependency of inhibition of alpha7 nicotinic receptors and tonically active N-methyl-D-aspartate receptors by endogenously produced kynurenic acid in the brain

Abstract

In the mouse hippocampus normal levels of kynurenic acid (KYNA), a neuroactive metabolite synthesized in astrocytes primarily by kynurenine aminotransferase II (KAT II)-catalyzed transamination of L-kynurenine, maintain a degree of tonic inhibition of α7 nicotinic acetylcholine receptors (nAChRs). The present in vitro study was designed to test the hypothesis that α7 nAChR activity decreases when endogenous production of KYNA increases. Incubation (2-7 h) of rat hippocampal slices with kynurenine (200 μM) resulted in continuous de novo synthesis of KYNA. Kynurenine conversion to KYNA was significantly decreased by the KAT II inhibitor (S)-(-)-9-(4-aminopiperazine-1-yl)-8-fluoro-3-methyl-6-oxo-2,3,5,6-tetrahydro-4H-1-oxa-3a-azaphenalene-5carboxylic acid (BFF122) (100 μM) and was more effective in slices from postweaned than preweaned rats. Incubation of slices from postweaned rats with kynurenine inhibited α7 nAChRs and extrasynaptic N-methyl-D-aspartate receptors (NMDARs) on CA1 stratum radiatum interneurons. These effects were attenuated by BFF122 and mimicked by exogenously applied KYNA (200 μM). Exposure of human cerebral cortical slices to kynurenine also inhibited α7 nAChRs. The α7 nAChR sensitivity to KYNA is age-dependent, because neither endogenously produced nor exogenously applied KYNA inhibited α7 nAChRs in slices from preweaned rats. In these slices, kynurenine-derived KYNA also failed to inhibit extrasynaptic NMDARs, which could, however, be inhibited by exogenously applied KYNA. In slices from preweaned and postweaned rats, glutamatergic synaptic currents were not affected by endogenously produced KYNA, but were inhibited by exogenously applied KYNA. These results suggest that in the mature brain α7 nAChRs and extrasynaptic NMDARs are in close apposition to KYNA release sites and, thereby, readily accessible to inhibition by endogenously produced KYNA.

Fig. 1.

Kynurenine increases the production of KYNA in rat hippocampal slices in vitro. Seven hippocampal slices were incubated in 3 ml of ACSF containing either LK (200 μM) alone or the admixture of LK (200 μM) with BFF122 (100 μM). Concentrations of KYNA were measured in ACSF sampled at different times after the start of the incubation and expressed as femtomole of KYNA per milligram of protein. The total protein content of hippocampal slices was determined using the BCA protein assay. Data points and error bars represent mean and S.E.M., respectively, obtained from seven experiments using kynurenine-incubated slices from three P30 rats, 13 experiments using kynurenine-incubated slices from three P10 rats, and five experiments using kynurenine-plus-BFF122-incubated slices from two P30 rats. *, p < 0.01; **, p < 0.001; ***, p < 0.0001, all compared with kynurenine P30 rats by one-way ANOVA followed by Bonferroni comparison.

Fig. 2.

Effects of kynurenine on α7 nAChR currents in rat hippocampal slices. A, sample recordings of whole-cell inward currents evoked by application of 10 mM choline to CA1 SRIs voltage-clamped at −60 mV. The agonist was applied via a U-tube for 12 s (indicated by bars at top of traces). Samples are representative of many such recordings in the two groups of slices at four age groups. Control represents slices incubated in LK-free ACSF. LK200 represents slices incubated in ACSF containing 200 μM LK. Incubations lasted 2 to 7 h. In addition, 200 μM LK was continuously present in the ACSF during the entire recording session in the LK group. Calibration in P30 applies to all traces. B, graph represents the peak amplitude of choline-evoked inward currents recorded from neurons in slices incubated in LK-free and LK-containing ACSF. The preweaned group consisted of animals from P10 and P18, and the postweaned group consisted of animals from P23 and P35. Graph and error bars represent mean and S.E.M., respectively, of results obtained from the numbers of neurons presented in parentheses. **, p < 0.0001, compared with preweaned controls; ***, p < 0.0001, compared with postweaned controls by one-way ANOVA followed by Bonferroni comparison. C, plot of the peak amplitude of choline-induced currents versus LK incubation time. Each symbol represents a neuron. The solid line represents the linear regression of the data points.

Fig. 3.

Effects of kynurenine on α7 nAChR-dependent GABAergic PSCs in rat hippocampal slices. A, sample recordings of outward-going GABAergic PSCs obtained from SRIs during U-tube application of choline (10 mM) to slices from preweaned (P10) and postweaned (P30) rats. Holding potential = 0 mV. Control slices were incubated in ACSF, whereas LK slices were incubated in LK (200 μM)-containing ACSF. Incubations lasted 2 to 7 h. B, graph represents the net charge of choline-induced GABAergic PSCs recorded from several neurons in the absence and presence of LK (200 μM). Data are presented as mean and S.E.M. of results obtained from the number of neurons shown in parentheses. **, p < 0.02, compared with P30 controls, by one-way ANOVA followed by Bonferroni post hoc test.

Fig. 4.

Kynurenine suppresses α7 nAChR-mediated responses in human cerebral cortical slices. A, neurolucida drawing of a biocytin-filled interneuron in a human lateral cortical slice. B, sample recording of a whole-cell current recorded at −60 mV during U-tube application of choline (10 mM) to the interneuron shown in A. C and D, sample recordings of choline (10 mM)-induced GABA PSCs obtained from interneurons voltage-clamped at 0 mV in cortical slices maintained in LK-free ACSF (C) or LK (200 μM)-containing ACSF (D). E and F, sample recordings of ACh (0.1 mM)-induced GABA PSCs obtained from neurons voltage-clamped at 0 mV in cortical slices maintained in LK-free ACSF (E) or LK (200 μM)-containing ACSF (F). In C to F, incubations lasted 3 h.

Fig. 5.

The KAT II inhibitor BFF122 (BFF) attenuates the inhibitory effect of kynurenine on α7 nAChR currents. A, graph shows the peak amplitude of choline (10 mM)-evoked type IA currents recorded under various experimental conditions. Graph and error bars represent mean and S.E.M., respectively, of data obtained from the numbers of neurons shown in parentheses. Hippocampal slices were incubated for 2 to 7 h in control ACSF or ACSF containing different test compounds. All data are from postweaned animals. *, p < 0.05; **, p < 0.01; ***, p < 0.001 according to one-way ANOVA followed by Bonferroni post hoc test. B, normalized peak amplitude of choline (10 mM)-evoked currents in three of the experimental groups depicted in A. Results obtained in the presence of LK were normalized to those obtained under control conditions. Results obtained in the presence of BFF122 + kynurenine were normalized to those obtained in the presence of BFF122 alone, and results obtained in the presence of TFA + LK were normalized to those obtained in the presence of TFA alone. ***, p < 0.0001 compared with LK by one-way ANOVA followed by Bonferroni comparison.

Fig. 6.

Age and concentration dependencies of KYNA-induced inhibition of α7 nAChR currents in rat hippocampal slices. A, graph of the peak amplitude of choline (10 mM)-evoked inward currents in control slices and slices incubated with KYNA (2–200 μM) for 2 to 7 h. Slices were obtained from preweaned (P10) and postweaned (P24) rats. B, the peak amplitude of choline (10 mM)-evoked currents recorded in the presence of KYNA or LK was normalized to that recorded in the absence of the two test compounds. The mean peak amplitude recorded from neurons in slices incubated with control ACSF was taken as 100% for each age group and used to normalize the peak amplitude of currents recorded after 2- to 7-h incubation of the slices with KYNA or LK. Graph and error bars represent mean and S.E.M., respectively, of results obtained from the numbers of neurons in parentheses. Results are significantly different from control according to one-way ANOVA followed by Dunnett's post hoc test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 7.

Comparison of the effects of kynurenine and exogenously applied KYNA on AMPA EPSCs recorded from CA1 SRIs in hippocampal slices from postweaned rats. Slices were incubated with ACSF alone or in the presence of various test compounds for 2 to 7 h and superfused with the same chemicals during the recordings. A, graphs represent the frequency, peak amplitude, 10 to 90% rise time, and decay-time constant of spontaneously occurring AMPA EPSCs recorded in the absence or in the presence of LK (200 μM), BFF122 (100 μM), or LK + BFF122. B, graphs represent the frequency, peak amplitude, 10 to 90% rise time, and decay-time constant of spontaneously occurring AMPA EPSCs recorded in the absence or presence of KYNA (100 or 200 μM) or APV (50 μM). Graph and error bars are mean and S.E.M., respectively, of the results obtained from the numbers of neurons in parentheses. *, p < 0.05; **, p < 0.01 compared with respective control by one-way ANOVA followed by Bonferroni post hoc test.

Fig. 8.

Effects of kynurenine and KYNA on NMDA currents and ACh-evoked EPSCs in hippocampal slices from rats at different ages. ACh (0.1 mM)-induced NMDA EPSCs (type III nAChR response) or NMDA (50 μM)-induced currents were recorded from CA1 SRIs at +40 mV. A 12-s pulse of ACh or a 30-s pulse of NMDA (no added glycine) was used. Hippocampal slices were incubated with ACSF containing no test compound or LK (200 μM) for 2 to 7 h. In experiments with KYNA, control responses recorded from any given neuron were compared with responses recorded from the same neuron at 10 min after the start of bath application of KYNA (200 μM). The net charge was calculated for the duration of the pulse. Graph and error bars are mean and S.E.M., respectively, of results obtained from the numbers of neurons in parentheses. *, p < 0.05; **, p < 0.01 compared with control by paired t test.

Fig. 9.

Kynurenine and KYNA suppress tonic NMDA currents in rat hippocampal slices. A, sample recordings obtained from CA1 SRIs under the whole-cell patch configuration at −60 mV illustrating baseline current fluctuations under various conditions. B, graphs represent the percentage change in the baseline current S.D. recorded from SRIs in the nominal absence of Mg²⁺ under control condition and in the presence of LK (200 μM) or KYNA (100 or 200 μM). Baseline current S.D. in nominally Mg²⁺-free ACSF is expressed as percentage of that in Mg²⁺-containing ACSF in all groups. Whole-cell currents were recorded from CA1 SRI of hippocampal slices from preweaned (P10) or postweaned (P24) rats. Slices were incubated for 2 to 7 h with ACSF containing LK (200 μM) or superfused for 10 min with KYNA (100 or 200 μM)-containing ACSF. Graph and error bars represent mean and S.E.M., respectively, of results obtained from the numbers of neurons in parentheses. *, p < 0.05; ***, p < 0.001 compared with control, by one-way ANOVA followed by Bonferroni comparison.

Fig. 10.

Schematic representation of the relative locations of KYNA-sensitive targets. This simplified scheme illustrates the role of astrocyte-derived KYNA in selectively modulating the activity of α7 nAChRs and tonically active NMDARs in a CA1 SRI. An axon from a glutamatergic neuron bearing α3β4β2 nAChRs and an axon from a cholinergic neuron are shown synapsing onto the CA1 SRI. Surrounding these synaptic contacts are astrocytes, where KAT II catalyzes the transamination of kynurenine into KYNA. Results presented in this study support the contention that exposure to kynurenine increases the astrocytic neosynthesis of KYNA, which is, in turn, released at sites in close apposition to α7 nAChRs and tonically active (extrasynaptic) NMDARs. The distance between the KYNA-release sites and synaptically located NMDARs and AMPA receptors (AMPAR) spares these receptors from inhibition by endogenously produced KYNA. In such tripartite synapses made up of astrocytes in addition to presynaptic and postsynaptic neurons, neuronal activity is ultimately modulated by astrocyte-derived KYNA.