mGluR1, but not mGluR5, activates feed-forward inhibition in the medial prefrontal cortex to impair decision making

Abstract

Cognitive flexibility depends on the integrity of the prefrontal cortex (PFC). We showed previously that impaired decision making in pain results from amygdala-driven inhibition of medial PFC neurons, but the underlying mechanisms remain to be determined. Using whole cell patch clamp in rat brain slices and a cognitive behavioral task, we tested the hypothesis that group I metabotropic glutamate receptors (mGluRs) activate feed-forward inhibition to decrease excitability and output function of PFC pyramidal cells, thus impairing decision making. Polysynaptic inhibitory postsynaptic currents (IPSCs) and monosynaptic excitatory postsynaptic currents (EPSCs) were evoked in layer V pyramidal cells by stimulating presumed amygdala afferents. An mGluR1/5 agonist [(S)-3,5-dihydroxyphenylglycine, DHPG] increased synaptic inhibition more strongly than excitatory transmission. The facilitatory effects were blocked by an mGluR1 [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid, LY367385], but not mGluR5, antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine. IPSCs were blocked by bicuculline and decreased by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX). Facilitation of synaptic inhibition by DHPG was glutamate driven because it was blocked by NBQX. DHPG increased frequency but not amplitude of spontaneous IPSCs; consistent with action potential-dependent synaptic inhibition, tetrodotoxin (TTX) prevented the facilitatory effects. DHPG decreased synaptically evoked spikes (E-S coupling) and depolarization-induced spiking [frequency-current (f-I) relationship]. This effect was indirect, resulting from glutamate-driven synaptic inhibition, because it persisted when a G protein blocker was included in the pipette but was blocked by GABA(A) receptor antagonists and NBQX. In contrast, DHPG increased E-S coupling and f-I relationships in mPFC interneurons through a presynaptic action, further supporting the concept of feed-forward inhibition. DHPG also impaired the ability of the animals to switch strategies in a decision-making task; bicuculline restored normal decision making, whereas a GABA(A) receptor agonist (muscimol) mimicked the decision-making deficit. The results show that mGluR1 activates feed-forward inhibition of PFC pyramidal cells to impair cognitive functions.

Fig. 1.

Polysynaptic inhibitory and monosynaptic excitatory transmission in the medial prefrontal cortex (PFC). A: stimulation and recording sites in a coronal brain slice containing the medial PFC, consisting of infralimbic (IL) and prelimbic (PL) cortical areas. Level is 3.2 mm anterior to bregma. B: course of afferent fibers in the medial PFC labeled anterogradely by fluorescent dye injection into the basolateral amygdala (BLA). Brain slices obtained 10–12 days after BLA injection show fluorescent labeling of fiber bundles that divide to reach layers II and V of IL and PL (Ji et al. 2010). No cell bodies are stained in layer V (arguing against retrograde labeling). C: individual pyramidal cell visually identified with infrared differential interference contrast videomicroscopy. D: whole cell patch-clamp recordings of excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) evoked by stimulating presumed BLA afferents with minimal stimulus intensities near threshold. Individual traces recorded at −70 mV (downward deflections, EPSCs) or at 0 mV (upward deflections, IPSCs). E: monosynaptic EPSCs, but not polysynaptic IPSCs, follow high-frequency stimulation (20 Hz) reliably with constant latency. F: distribution of latencies measured from the stimulus artifact to the onset of EPSCs and IPSCs in an individual pyramidal cell. G: average latencies of IPSCs and EPSCs (n = 6 neurons). Latencies were measured from stimulus artifact to onset of synaptic current and from stimulus artifact to peak amplitude. Bar histograms show means ± SE. Latencies of IPSCs were significantly longer. ***P < 0.05 (paired t-test).

Fig. 2.

(S)-3,5-dihydroxyphenylglycine (DHPG) increases inhibitory synaptic transmission more strongly than excitatory transmission. Whole cell voltage-clamp recordings of visually identified prelimbic layer V pyramidal cells as in Fig. 1. A: DHPG (1 μM) increased evoked IPSCs (recorded at 0 mV). B: DHPG also increased evoked EPSCs (recorded at −70 mV). A and B: individual traces are averages of 8–10 PSCs. Scale bars = 100 pA, 100 ms. C and D: DHPG (1 μM) significantly increased input-output functions of IPSC peak amplitude (n = 15 neurons, 2-way ANOVA, F_1,308 = 63.24, P < 0.0001, C) and area under the curve (2-way ANOVA, F_1,308 = 87.20, P < 0.0001, D). E and F: DHPG (1 μM) also significantly increased input-output functions of EPSC peak amplitude (n = 9 neurons, 2-way ANOVA, F_1,176 = 24.28, P < 0.0001, E) and area under the curve (2-way ANOVA, F_1,308 = 108.67, P < 0.0001, F). C–F: *P < 0.05, **P < 0.01, ***P < 0.001 (Bonferroni posttests). G and H: difference between DHPG and predrug values of amplitude (G) and total charge (H) was significantly greater for IPSCs compared with EPSCs (P < 0.01–0.001, unpaired t-test). Stimulation intensity was 700 μA. C–H: symbols and bar histograms show means ± SE.

Fig. 3.

(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385, LY), but not 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), antagonizes the effects of DHPG on inhibitory and excitatory transmission. Whole cell voltage-clamp recordings of visually identified prelimbic layer V pyramidal cells. A: LY367385 (10 μM) inhibited the increase of IPSCs (recorded at 0 mV) and EPSCs (recorded at −70 mV) induced by DHPG (1 μM). B: MTEP (10 μM) had no effect on the DHPG-induced increase of IPSCs and EPSCs. A and B: individual traces are averages of 8–10 PSCs. Scale bars = 100 pA, 100 ms. C and D: effects of DHPG (1 μM) alone and in the presence of LY367385 (10 μM, n = 5) or MTEP (10 μM, n = 13) on IPSC total charge normalized to predrug control values (set to 100%). E and F: effects of DHPG (1 μM) alone and in the presence of LY367385 (10 μM, n = 5) or MTEP (10 μM, n = 7) on EPSC total charge normalized to predrug control values (set to 100%). Bar histograms show means ± SE. *P < 0.05, **P < 0.01 compared with predrug; #P < 0.05, compared with DHPG; ns, not significant, compared with DHPG (Tukey's multiple-comparison tests).

Fig. 4.

2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX) blocks the facilitatory effect of DHPG on inhibitory synaptic transmission. Whole cell voltage-clamp recordings of visually identified prelimbic layer V pyramidal cells. A: diagram illustrates our hypothesis that DHPG (through metabotropic glutamate receptor 1, mGluR1) activates feed-forward inhibition of medial PFC cells through a mechanism that involves non-N-methyl-d-aspartate (NMDA) (3-hydroxy-5-methylisoxazole propionic acid, AMPA) and GABA_A receptors. Squares indicate receptors. B: individual traces (averages of 8–10 IPSCs recorded at 0 mV) show that IPSCs were blocked by a GABA_A receptor antagonist (bicuculline, 10 μM). C: individual traces (averages of 8–10 IPSCs) show that IPSCs were inhibited by a non-NMDA (AMPA) receptor antagonist (NBQX, 10 μM). DHPG (1 μM) coapplied with NBQX had no effect. D: NBQX inhibited GABAergic IPSCs significantly (n = 5, 2-way ANOVA, F_1,88 = 80.14, P < 0.0001) and blocked the effect of DHPG (n = 5, 2-way ANOVA, F_1,88 = 0.95, P > 0.05, compared with NBQX alone). Symbols show means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

DHPG-induced synaptic inhibition is action potential dependent. Whole cell voltage-clamp recordings of visually identified prelimbic layer V pyramidal cells. A: diagram illustrates the hypothesis that DHPG activates feed-forward inhibition through an action potential-dependent mechanism rather than a site of action on the GABAergic terminal. B: individual traces show spontaneous IPSCs before (predrug) and during DHPG application (1 μM). C: DHPG (1 μM) increased frequency but not amplitude of spontaneous IPSCs (sIPSC) (n = 10 neurons, paired t-test, P < 0.01). Bar histograms show averaged values before (white bars) and during DHPG application. D: individual traces show miniature IPSCs (mIPSC) in tetrodotoxin (TTX) (1 μM) before (predrug) and during application of DHPG (1 μM). E: in the presence of TTX (1 μM) DHPG had no effect (n = 5 neurons). Bar histograms show averaged values before (white bars) and during application of DHPG. **P < 0.01.

Fig. 6.

DHPG-induced synaptic inhibition inhibits pyramidal output (synaptically evoked spiking). Whole cell current-clamp recordings were made of visually identified prelimbic layer V pyramidal cells. A: diagram illustrates our hypothesis that feed-forward inhibition decreases pyramidal output through an action potential-dependent GABAergic mechanism. B: individual traces (10 each) show excitatory postsynaptic potentials (EPSPs) and spikes evoked with near-threshold stimulus intensity from a holding potential of −60 mV before (predrug) and during DHPG (1 μM) and in the presence of DHPG together with bicuculline (10 μM). C, left: bar histograms show average number of evoked spikes per synaptic stimulation in 10 successive trials; right: probability of synaptically evoked spikes were calculated as follows: (number of trials with evoked spikes)/(number of trials). DHPG (1 μM) (n = 5 neurons); DHPG together with bicuculline (10 μM), n = 5 neurons. D and E: inhibitory effects of DHPG persist when guanosine 5-O-(2-thiodiphosphate) (GDP-β-S) (1 mM) is included in the patch pipette (n = 5 neurons). F and G: membrane hyperpolarization by DHPG is blocked with TTX (1 μM; n = 5 neurons) and bicuculline (10 μM, n = 7). Mean resting membrane potential, 65 ± 1 mV. H and I: bicuculline (10 μM, n = 5) increases the number of synaptically evoked spikes (left) and spiking probability (right); same display as in C. J and K: picrotoxin (30 μM, n = 5) also increases synaptically evoked spiking (see C and I). Scale bars in B, D, H, and J = 25 mV, 5 ms; scale bars in F = 5 mV, 60 s. Bar histograms show means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 (E, I, and K, paired t-test; G, ANOVA with Bonferroni posttests, compared with DHPG).

Fig. 7.

DHPG-induced synaptic inhibition inhibits pyramidal output (depolarization-induced spiking). Whole cell current-clamp recordings of visually identified prelimbic layer V pyramidal cells. Action potentials were generated by direct intracellular current injections (500 ms) of increasing magnitude (in 50-pA steps) from a holding potential of −60 mV. Right: original voltage traces showing action potentials evoked in individual cells by current injections of 0 pA and 300 pA. Left: graphs showing input-output functions (f-I relationships) averaged for each sample of neurons. A: DHPG (1 μM) decreased the input-output function significantly (n = 28 neurons, 2-way ANOVA, F_1,432 = 257.01, P < 0.0001). ***P < 0.001 (Bonferroni posttests). B: when GDP-β-S (1 mM) was included in the patch pipette (predrug), DHPG still had inhibitory effects (n = 5 neurons, 2-way ANOVA, F_1,64 = 16.84, P < 0.001). *P < 0.05 (Bonferroni posttests). C: bicuculline (10 μM) itself did not affect neuronal excitability but blocked the inhibitory effect of DHPG (n = 7 neurons, 2-way ANOVA, F_1,96 = 0.28, P > 0.05). D: NBQX (10 μM) also had no effect on action potential firing but blocked the effect of DHPG (n = 6, 2-way ANOVA, F_1,80 = 0.57, P > 0.05). Symbols show means ± SE.

Fig. 8.

DHPG increases output of interneurons through a presynaptic mechanism. Whole cell current-clamp recordings were made of fast-spiking nonaccommodating interneurons with fast afterhyperpolarization (Markram et al. 2004; Zhou and Hablitz 1996) that were visually identified as nonpyramidal cells in layer V of the prelimbic cortex. A: DHPG (1 μM) increased the frequency-current (f-I) function of PFC interneurons significantly (n = 5 neurons, 2-way ANOVA, F_1,64 = 210.94, P < 0.0001). Input-output functions were averaged for each sample of neurons (mean ± SE). B: action potentials evoked in an individual interneuron by current injections of 0 pA and 300 pA. Action potentials were generated by direct intracellular current injections (500 ms) of increasing magnitude (in 50-pA steps) from a holding potential of −65 mV. C: individual traces show miniature EPSCs in TTX (1 μM) before (predrug) and during application of DHPG (1 μM). D and E: DHPG decreased frequency (n = 5, paired t-test, P < 0.05) but not amplitude of mEPSCs in mPFC interneurons, consistent with a presynaptic site of action. Bar histograms show means ± SE. ***P < 0.001, *P < 0.05.

Fig. 9.

DHPG acts presynaptically to increase excitatory transmission onto pyramidal cells. Whole cell voltage-clamp recordings of visually identified prelimbic layer V pyramidal cells. A: diagram illustrates our hypothesis that mGluR1 acts presynaptically to regulate glutamatergic transmission onto interneurons as well as pyramidal cells. B: individual traces show mEPSCs (in TTX, 1 μM) recorded before and during DHPG (1 μM). C and D: cumulative distribution analysis (see methods) of mEPSC frequency (C) and amplitude (D). Bar histograms show mean frequency and amplitude averaged for the sample of neurons (n = 5) before (predrug) and during DHPG. DHPG (1 μM) increased frequency but not amplitude of mEPSCs significantly (cumulative frequency distribution, P < 0.001, Kolmogorov-Smirnov test; mean frequency, P < 0.05, paired t-test, n = 5 neurons). E: individual traces (averages of 3–4 EPSCs) show that DHPG (1 μM) decreased paired-pulse facilitation of evoked EPSCs. F: paired-pulse ratio of peak amplitudes of 2 consecutive EPSCs (EPSC2/EPSC1) was measured at different interstimulus intervals under control conditions (predrug) and in the presence of DHPG (1 μM). The overall effect of DHPG was significant (n = 5 neurons, 2-way ANOVA, F_1,40 = 16.77, P < 0.001). Bonferroni posttests showed a significant difference for the 20 ms interstimulus interval. *P < 0.05.

Fig. 10.

DHPG impairs decision making through a GABAergic mechanism in the PFC. Animals chose between low-risk (1 food pellet in 9 of 10 visits) and high-risk (3 pellets in 3 of 10 visits) levers in 90 consecutive trials (see methods). Symbols (mean ± SE) show the preference index [(low-risk − high-risk choices)/number of trials] averaged for 10 consecutive trials of animals in 3 experimental groups. A: normal animals (n = 6) switched from high- to low-risk choices. Rats in which DHPG (100 μM) was administered into the medial PFC by microdialysis (see methods) failed to switch strategies (n = 5). Their preference index was significantly different from that of normal rats (2-way ANOVA, F_1,81 = 28.16, P < 0.0001). Coapplication of bicuculline (1 mM) with DHPG restored normal decision making (n = 5). Preference index was not significantly different from that of normal rats (2-way ANOVA, F_1,81 = 2.38, P > 0.05). Administration of a GABA_A receptor agonist (muscimol, 0.5 mM, n = 5) (Nattie and Li 2008) into the medial PFC mimicked the effect of DHPG and resulted in a preference index that was significantly different from that of normal animals (F_1,81 = 32.31, P < 0.0001 compared with normal). **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with normal (Bonferroni posttests). B: placement control for drug diffusion. Administration of DHPG into the anterior cingulate cortex dorsal to the prelimbic PFC (n = 5) did not impair the ability to switch strategies, which is reflected in the significant change of the preference index compared with the initial preference index value (F = 6.06, P < 0.0001 repeated-measures ANOVA). *P < 0.05, ***P < 0.001 compared with initial preference index (Dunnett's posttests).