Mechanisms underlying prelimbic prefrontal cortex mGlu3/mGlu5-dependent plasticity and reversal learning deficits following acute stress

Abstract

Stress can precipitate or worsen symptoms of many psychiatric illnesses. Dysregulation of the prefrontal cortex (PFC) glutamate system may underlie these disruptions and restoring PFC glutamate signaling has emerged as a promising avenue for the treatment of stress disorders. Recently, we demonstrated that activation of metabotropic glutamate receptor subtype 3 (mGlu₃) induces a postsynaptic form of long-term depression (LTD) that is dependent on the activity of another subtype, mGlu₅. Stress exposure disrupted this plasticity, but the underlying signaling mechanisms and involvement in higher-order cognition have not yet been investigated. Acute stress was applied by 20-min restraint and early reversal learning was evaluated in an operant-based food-seeking task. We employed whole-cell patch-clamp recordings of layer 5 prelimbic (PL)-PFC pyramidal cells to examine mGlu₃-LTD and several mechanistically distinct mGlu₅-dependent functions. Acute stress impaired both mGlu₃-LTD and early reversal learning. Interestingly, potentiating mGlu₅ signaling with the mGlu₅ positive allosteric modulator (PAM) VU0409551 rescued stress-induced deficits in both mGlu₃-LTD and reversal learning. Other aspects of PL-PFC mGlu₅ function were not disrupted following stress; however, signaling downstream of mGlu₅-Homer interactions, phosphoinositide-3-kinase (PI3K), Akt, and glycogen synthase kinase 3β was implicated in these phenomena. These findings demonstrate that acute stress disrupts early reversal learning and PL-PFC-dependent synaptic plasticity and that potentiating mGlu₅ function can restore these impairments. These findings provide a framework through which modulating coordinated mGlu₃/mGlu₅ signaling may confer benefits for the treatment of stress-related psychiatric disorders.

Keywords: Prelimbic prefrontal cortex; Reversal learning; Stress; Synaptic plasticity; mGlu(3); mGlu(5).

Figure 1.. Potentiating mGlu₅ function rescues stress-induced deficits in mGlu₃/mGlu₅ plasticity.

(a) Representative experiment showing that LY379268, an orthosteric agonist for metabotropic glutamate (mGlu) receptor subtype 3 (mGlu₃), induces long-term depression (LTD) of excitatory transmission onto pyramidal cells in the prelimbic prefrontal cortex (PL-PFC). (b) Summary of all control PL-PFC mGlu₃-LTD recordings (60.5 ± 4.9% baseline, n/N = 9/8 cells/mice). (c) Schematic, acute restraint stress was applied 30 minutes before mice were sacrificed for electrophysiology. (d) Acute restraint impairs the induction of ex vivo LTD (93.4 ± 8.0% baseline, n/N = 7/6). (e) In slices from stress-exposed mice, application of an mGlu₅ positive allosteric modulator, VU0409551, restores the expression of LTD (69.4 ± 5.6% baseline, n/N = 6/5). A one-way ANOVA revealed a main effect of treatment (F_1,2 = 8.337; p = 0.0025) and Bonferonni post-tests confirmed the stress-induced impairment and rescue with VU0409551 bath application. (f) VU0409551 does not alter excitatory transmission by itself in slices from stress-exposed mice. (100.6 ± 13.8% baseline, n/N = 5/4). EPSC, excitatory postsynaptic current; LTD, long-term depression; mGlu₃ and mGlu₅, metabotropic glutamate receptor subtype 3 and 5; PL-PFC, prelimbic prefrontal cortex.

Figure 2.. Several mGlu₅-dependent functions remain intact following acute stress.

(a) Schematic displaying acute stress exposure paradigm. Mice underwent 20 minutes of restraint stress and were sacrificed for electrophysiology 30 minutes later. (b) Inward currents induced by a threshold and high concentration of the orthosteric agonist, DHPG, were acquired in the presence of the metabotropic glutamate (mGlu) receptor subtype 1 (mGlu₁) negative allosteric modulator VU0469650 and tetrodotoxin. These mGlu₅-mediated currents were not different between controls (13.1 ± 3.0, 36.7 ± 8.7 pA, n/N = 7/3 cells/mice) and the restraint stress group (18.8 ± 3.9, 38.0 ± 7.1 pA, n/N = 5/3, 9/5). (c/d) mGlu₅-dependent LTD was elicited with the muscarinic agonist carbachol (CCH). CCH-LTD was comparable between control (80.8 ± 6.5% baseline, n/N = 8/5) and restraint stress groups (78.6 ± 7.8% baseline, n/N = 6/3). (e) The charge transfer through small conductance potassium (SK) channels was determined as the charge transfer of the medium afterhyperpolarization (Q_AHP) following increasing voltage steps (F_3,33 = 45.63, p < 0.0001). Baseline SK channel function was not altered following restraint stress (F_1,11 = 0.36, n.s.). (f) mGlu₅ activation inhibited SK channel function to a similar degree in control (21.7 ± 7.8% inhibition, n/N = 10/4) and restraint stress mice (21.7 ± 6.9% inhibition, n/N = 6/2). (g) Control experiments displaying that the selective SK channel inhibitor apamin completely blocked Q_AHP (103.1 ± 3.9% inhibition, n/N = 6/3, *: p < 0.0001 one-sample t-test vs. 0% inhibition), while VU0469650 exerted no effect by itself (−13.5 ± 8.2% inhibition, n/N = 5/3). CCH, carbachol; EPSC, excitatory postsynaptic current; LTD, long-term depression; mGlu₁ and mGlu₅, metabotropic glutamate receptor subtype 1 and 5; SK, small conductance potassium; Q_AHP, charge transfer of the medium afterhyperpolarization.

Figure 3.. mGlu₃-LTD proceeds through PI3K-Akt signaling pathway.

(a) The divalent ion chelator EGTA was added to the patch pipette to quench Ca²⁺ signaling in the postsynaptic cell. This manipulation had no effect on the expression of metabotropic glutamate receptor subtype 3 (mGlu₃)-dependent long-term depression (LTD) (62.1 ± 6.9% baseline, n/N = 7/4 cells/mice). Black lines in panels 3a-4d represent data from control experiments displayed in panel 1b. (b) The divalent ion chelator BAPTA was added to the patch pipette and did not affect mGlu₃-LTD (59.9 ± 4.9% baseline, n/N = 5/3). (c) The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 impaired the expression of mGlu₃-LTD (85.7 ± 5.1% baseline, n/N = 8/6). (d) The Akt inhibitor Akti-1/2 blocked mGlu₃-LTD (89.6 ± 11% baseline, n/N = 5/4). EPSC, excitatory postsynaptic current; LTD, long-term depression; mGlu₃, metabotropic glutamate receptor subtype 3; PI3K, phosphoinositide 3-kinase.

Figure 4.. mGlu₃-LTD is modulated by mGlu₅-Homer interactions and Glycogen synthase kinase 3.

(a) Inclusion of a peptide that blocks the interaction between metabotropic glutamate receptor subtype 5 (mGlu₅) and Homer proteins (mGlu₅-C-ter) in the patch pipette enhances mGlu₃-LTD (49.0 ± 7.5% baseline, n/N = 4/2 cells/mice) relative to a control peptide (mGlu₅-mut) that does not impair the mGlu₅-Homer interaction (73.2 ± 4.3% baseline, n/N = 5/4). (b) Whole-cell infusion of mGlu₅-C-ter generates LTD following threshold application of LY379268 (67.8 ± 5.6% baseline, n/N = 6/4), whereas inclusion of mGlu₅-mut in the pipette has no effect (93.6 ± 9.8% baseline, n/N = 6/4). (c) Summary of the last 10 minutes of the recordings from the timecourse experiments (*: p < 0.05, **: p < 0.01, t-tests). (d) The mechanistic target of rapamycin inhibitor KU-0063794 does not affect mGlu₃-LTD (69.2 ± 5.2% baseline, n/N = 7/5). Black lines in panels 4d-5f denote control experiments from figure 1b. (e) Blocking protein translation with anisomycin does not impair mGlu₃-LTD (62.7 ± 6.4% baseline, n/N = 6/6). (f) The glycogen synthase kinase 3 inhibitor CHIR 99203 blocks the induction of LTD (89.6 ± 11.0% baseline, n/N = 5/5). EPSC, excitatory postsynaptic current; LTD, long-term depression; mGlu₅, metabotropic glutamate receptor subtype 5.

Figure 5.. Mechanisms of mGlu₃-LTD in the prelimbic prefrontal cortex.

(a) Long-term depression (LTD) of excitatory transmission in the prelimbic prefrontal cortex (PL-PFC) was induced by bath application of LY379268, an orthosteric agonist at metabotropic glutamate (mGlu) receptor subtype 3 (mGlu₃). In previous publications (Di Menna et al, 2018; Joffe et al, 2017), we have demonstrated that this LTD requires the activity of mGlu₃ and mGlu₅. Here, we demonstrate that PL-PFC mGlu₃-LTD is not impaired by manipulations that sequester intracellular Ca²⁺ (EGTA and BAPTA) or block the mechanistic target of rapamycin and protein translation (KU-0063794 and anisomycin). Instead, PL-PFC mGlu₃-LTD is blocked by a Phosphoinositide-3-kinase (PI3K) inhibitor (LY294002), an Akt inhibitor (Akti-1/2), and a Glycogen synthase kinase 3 inhibitor (CHIR 99201). The number in each bar represents the number of cells per experiment. A one-way ANOVA revealed a main effect of treatment (F_1,7 = 3.957; p = 0.0019). Grey bars denote no significant difference from Control. Black bars denote p < 0.05, Bonferonni post-test vs. Control. (b) Conclusions drawn from the present studies are displayed in bold and findings from two previous papers are italicized. While activation of mGlu₃ potentiates mGlu₅-mediated Ca²⁺ mobilization, the present findings suggest that is not related to LTD. Instead, mGlu₃-LTD is modulated by interactions between mGlu₅ and Homer proteins and requires signaling through PI3K, Akt, and GSK3. This signaling culminates with the dynamin-dependent internalization of AMPA receptors and a long-term decrease in postsynaptic quantal size and content. EPSC, excitatory postsynaptic current; GSK3β, glycogen synthase kinase 3β LTD, long-term depression; mGlu₃ and mGlu₅, metabotropic glutamate receptor subtype 3 and 5; PI3K, phosphoinositide 3-kinase; PL-PFC, prelimbic prefrontal cortex.

Figure 6.. Acute restraint stress impairs early reversal learning.

(a) Schematic. Mice were trained in an operant conditioning apparatus to holepoke on a continuous schedule of reinforcement for liquid food. On the day of testing only, some mice underwent a 20-minute restraint stress exposure. One hour after the stress terminated, mice underwent an operant test session where the active holepoke designation was reversed. (b) There was a main effect of stress (F_1,10 = 7.07, p < 0.03), session (F_6,60 = 18.62, p < 0.001), and an interaction (F_6,60 = 3.3, p < 0.01) on performance across training, reversal, and follow-up sessions. Post-tests revealed a strong trend towards a decrease in the number of correct holepoke responses on the day of the reversal (7 ± 3 vs. 24 ± 7 holepokes, N = 5–7 mice, 95% confidence interval of difference: [−35.6, 0.8] Bonferonni post-test). In addition, this deficit carried over until the following day, when the mice were not stressed before the initiation of the task (21 ± 7 vs. 49 ± 1 holepokes, ***: p < 0.001, Bonferonni post-test). (c) Stressed mice exhibited decreased accuracy during the reversal session (9.3 ± 2.2% vs. 26.8 ± 5.0% correct holepokes, **: p<0.01, t-test). (d) Stressed mice required more days to reach criteria following the reversal than controls (χ² = 5.4, df=1, p < 0.02, Mantel-Cox Test). (e) Stressed mice displayed a longer latency to retrieve the food reward than controls during the reversal session (2.4 ± 0.5 vs. 4.1 ± 0.5s, *: p<0.05, t-test).

Figure 7.. Potentiating mGlu₅ function rescues stress-induced deficit in early reversal learning.

(a) Schematic for behavioral experiments. Mice were trained in an operant conditioning apparatus to holepoke on a continuous schedule of reinforcement for liquid food. On the day of testing only, some mice underwent a 20-minute restraint stress exposure. One hour after the stress terminated, mice underwent an operant test session where the active holepoke designation was reversed. VU0409551 or vehicle was administered 30-minutes after the stress and before the reversal test session. (b) There was a trend effect of treatment (F_2,13 = 2.9, p < 0.1), a significant main effect of session (F_6,78 = 26.03, p < 0.001), and a significant interaction (F_12,78 = 2.5, p < 0.01) on performance across training, reversal, and post-test sessions. Post-tests revealed that mice exposed to acute restraint exhibited a decrease in the number of correct holepoke responses on the day of the holepoke reversal (0 ± 1 vs. 23 ± 7 holepokes, N = 4–6 mice, *: p<0.05, Bonferonni post-test vs. control/veh). In addition, this deficit carried over until the following day, when the mice were not stressed before the initiation of the task (14 ± 6 vs. 44 ± 4 holepokes, ***: p<0.001, Bonferonni post-test vs. control/veh). VU0409551 administration rescued the stress-induced deficit in reversal learning on both the day of reversal and the following day (20 ± 10 holepokes, N = 6, #: p<0.05, Bonferonni post-test vs. stress/veh). (c) Stressed mice exhibited decreased accuracy during the reversal session (0 ± 0.3% vs. 27.4 ± 3.3% correct holepokes, N = 4–6, *: p<0.05, Bonferonni post-test vs. control) that was rescued by pretreatment with VU0409551 (20 ± 8.5% correct holepokes, N = 6, #: p<0.05, Bonferonni post-test vs. stress). mGlu₃ and mGlu₅, metabotropic glutamate receptor subtype 3 and 5; veh, vehicle; VU551, VU0409551.