M1 Muscarinic Receptors Modulate Fear-Related Inputs to the Prefrontal Cortex: Implications for Novel Treatments of Posttraumatic Stress Disorder

Abstract

Background: The prefrontal cortex (PFC) integrates information from multiple inputs to exert top-down control allowing for appropriate responses in a given context. In psychiatric disorders such as posttraumatic stress disorder, PFC hyperactivity is associated with inappropriate fear in safe situations. We previously reported a form of muscarinic acetylcholine receptor (mAChR)-dependent long-term depression in the PFC that we hypothesize is involved in appropriate fear responding and could serve to reduce cortical hyperactivity following stress. However, it is unknown whether this long-term depression occurs at fear-related inputs.

Methods: Using optogenetics with extracellular and whole-cell electrophysiology, we assessed the effect of mAChR activation on the synaptic strength of specific PFC inputs. We used selective pharmacological tools to assess the involvement of M₁ mAChRs in conditioned fear extinction in control mice and in the stress-enhanced fear-learning model.

Results: M₁ mAChR activation induced long-term depression at inputs from the ventral hippocampus and basolateral amygdala but not from the mediodorsal nucleus of the thalamus. We found that systemic M₁ mAChR antagonism impaired contextual fear extinction. Treatment with an M₁ positive allosteric modulator enhanced contextual fear extinction consolidation in stress-enhanced fear learning-conditioned mice.

Conclusions: M₁ mAChRs dynamically modulate synaptic transmission at two PFC inputs whose activity is necessary for fear extinction, and M₁ mAChR function is required for proper contextual fear extinction. Furthermore, an M₁ positive allosteric modulator enhanced the consolidation of fear extinction in the stress-enhanced fear-learning model, suggesting that M₁ positive allosteric modulators may provide a novel treatment strategy to facilitate exposure therapy in the clinic for the treatment of posttraumatic stress disorder.

Keywords: Fear extinction; M(1) muscarinic receptor; Positive allosteric modulator; Posttraumatic stress disorder; Prefrontal cortex; Synaptic plasticity.

Figure 1:. Muscarinic LTD in the PFC is Input-Specific.

Acute slices of the mouse PFC were prepared 4 weeks after AAV-CaMKIIa-ChR2-eYFP was injected into the vHipp (blue), BLA (green) or MDT (purple). (A) Electrical stimulation of PL layer II/III evoked field excitatory postsynaptic potentials (efEPSPs) recorded in layer V (inset, sample traces). Application of 10μM OxoM induces an acute depression followed by LTD of efEPSPs measured 55-59 min post-drug add. (53.49 ± 10.47%; n = 7) (B) Optical stimulation of afferents from vHipp-ChR2 injected mice with paired pulses of 470nm blue light (1ms pulse duration; 50ms interpulse interval) elicited ofEPSPs that also underwent induction of LTD following bath application of OxoM (10μM). (62.01 ± 9.50%; n = 7) (C) ofEPSPs evoked from stimulation of BLA-ChR2 afferents were also sensitive to OxoM (10μM) and expressed LTD. (65.61 ± 5.28%; n = 7) (D) ofEPSPs evoked in MDT-ChR2 mice exhibited a small acute depression in the presence of OxoM (10μM) but rapidly returned to baseline, not expressing LTD. (100.6 ± 3.72%; n = 6). Sample traces for A-D correspond to baseline (1) and grey shaded area (2). Scale bars: 0.2mV and 20ms. (E) Summary data of change in fEPSP amplitude for each input; 1 = baseline amplitude, 2 = amplitude at 55-59min post-drug add corresponding to the grey shaded regions in A-D. Paired student’s t-test: Electrical, vHipp *p < 0.05, BLA ***p < 0.001, MDT p = 0.778. (F) Summary data of fEPSP amplitude corresponding to grey shaded regions expressed as a percent of baseline compared across inputs. One-way ANOVA: F_3,23 = 6.228, p = 0.003. Bonferroni’s post-test: Electrical vs. MDT: ** p < 0.01, vHipp vs MDT and BLA vs MDT: * p < 0.05, Electrical vs. vHipp: p > 0.05; Electrical vs. BLA: p > 0.05; BLA vs. vHipp: p > 0.05.

Figure 2:. Input-Specific mAChR LTD is mediated by M₁ Receptors.

Recordings from vHipp-ChR2 or BLA-ChR2 injected mice. (A) In the constant presence of the selective M₁ antagonist VU0255035 (VU’035, 10μM), OxoM (10μM) induced an acute depression of ofEPSPs PL layer V evoked from vHipp afferents but mAChR LTD was blocked. (99.96 ± 7.67%; n = 5). (B) Bath application of the selective M₁ allosteric agonist VU0364572 (VU’572, 30μM) for 10 min also induces LTD of ofEPSPs elicited from vHipp afferent stimulation (69.48 ± 6.38%; n = 5). (C) Summary data for vHipp ofEPSP amplitude 55-59 min post-drug add. Unpaired student’s t-test, OxoM vs. OxoM + VU’035: * p < 0.05; paired student’s t-test comparing baseline to shaded area in B: &, p < 0.05. (D) LTD of ofEPSPs evoked from BLA-ChR2 expressing afferents in response to OxoM (10μM) was also blocked in the constant presence of VU’035 (83.86 ± 3.34%; n = 6). (E) Bath application of VU’572 for 10 minutes also induces LTD of ofEPSPs elicited from BLA afferent stimulation (61.71 ± 4.24%; n = 5). (F) Summary data for BLA ofEPSP amplitude 55-59 min post-drug add. Unpaired student’s t-test, OxoM vs. OxoM + VU’035: * p < 0.05; paired student’s t-test comparing baseline to shaded area in E: &, p < 0.05. Shaded time courses in A and D correspond to OxoM alone from Fig 1. Solid colored line represents mean ofEPSP amplitude and grey shaded region around line is ± SEM.

Figure 3:. vHipp-PFC mAChR LTD Requires Postsynaptic M₁ Receptors.

(A) Chrm1^loxP/loxP mice were injected with AAV-CaMKIIa-Cre-mCherry (white with blue outline throughout) or AAV-CaMKIIa-mCherry (red with blue outline throughout) into the PFC and co-injected with AAV-CaMKIIa-ChR2-eYFP into the vHipp. Recordings were performed 5-6 weeks post-injection. (B) OxoM (10μM) induced an inward current in neurons from control mCherry-infected mice (−88.55 ± 10.92 pA; n = 7) but failed to elicit an inward current in neurons from Cre-mCherry infected mice (−8.651 ± 6.06 pA; n = 6). (Student’s t-test, mCherry vs. Cre **** p < 0.0001). (C) OxoM (10μM) induced a significant increase in sEPSC frequency recorded before optical stimulation in mCherry neurons. (One-way repeated measures ANOVA, F_2,6 = 13.52, p < 0.001, Bonferroni’s post-test *** p < 0.001 baseline (BL) vs OxoM, n = 7). (D) Conversely, OxoM induced a significant decrease in sEPSC frequency in Cre-mCherry neurons. (One-way repeated measures ANOVA, F_2,4 = 11.49, p < 0.01, Bonferroni’s post-test * p < 0.05 BL vs OxoM, ** p < 0.01 BL vs LTD, n = 5). (E) A representative experiment for an mCherry-infected neuron (scale bar: 25pA and 25ms) and (F) a Cre-infected neuron (scale bar: 100pA and 25ms). (G) Summary time course for control mCherry mAChR LTD experiments. Bath application of OxoM (10μM) induced a long-term depression of oEPSCs evoked from vHipp-ChR2 terminals in mCherry-infected neurons (60.15 ± 6.67%; n = 7). (H) Summary time course for Cre LTD experiments. LTD of oEPSCs was attenuated in Cre-mCherry infected neurons (82.66 ± 6.13%; n = 6). In both G and H, light shaded areas correspond to the time at which Oxo-M sEPSC measurements were taken for C and D. Dark shaded areas correspond to the time at which LTD sEPSC measurements were taken for C and D and for quantification in I. (I) Summary data for oEPSC amplitude 40-49 min post-OxoM add. Unpaired student’s t-test, * p < 0.05.

Figure 4:. M₁ Receptor Function is Necessary for Contextual but not Cued Fear Extinction.

Effect of systemic M₁ antagonism on cued and contextual fear extinction in mice. (A) Schematic depicting the training and testing procedure used. Mice were conditioned in Context A with 5 mild footshocks, each preceded by a 30s tone. On day 2, mice were administered the M₁ antagonist VU0255035 (3, 10, 30 mpk, i.p.) or vehicle (20% β-cyclodextrin) 30 minutes before being exposed to a series of 12 tones in a novel Context B to assess extinction of auditory cued fear. On day 3, mice were placed back in Context B and exposed to 9 tones to assess consolidation of cued fear extinction. On day 4, mice were again administered VU0255035 or vehicle and placed in Context A for 12 minutes to assess contextual fear extinction. On day 5, mice were placed back in Context A for 3 minutes to assess contextual fear extinction consolidation. (B) At all doses VU0255035 had no effect on auditory cued fear extinction on extinction day 2 (two-way repeated-measures ANOVA, Effect of drug: F_3,35 = 0.960, p = 0.423; Effect of tone block: F_3,35 = 36.00, p < 0.0001; Interaction, F_9,35 = 1.787, p = 0.079) or on recall day 3 (one-way ANOVA, F_3,35 = 0.350, p = 0.789). Data for days 2 and 3 are binned by 3 tones and mice were excluded from analysis if baseline freezing was >30%. Bar graph depicts average % freezing to the first three tones on recall day 3, corresponding to the grey shaded box. (N, Veh = 13, 3 mpk = 5, 10 mpk = 12, 30 mpk = 9) (C) Systemic M₁ antagonism impairs within-session contextual fear extinction (two-way repeated-measures ANOVA, Effect of drug: F_3,39 = 3.663, p = 0.020; Effect of time block: F_3,39 = 12.56, p < 0.0001; Interaction: F_3,39 = 0.317, p = 0.968) and 30 mpk VU0255035 significantly impaired contextual extinction recall on day 5 (One-way ANOVA, F_3,39 = 5.177, p < 0.01; Bonferroni’s post-test, Veh vs. 30 mpk * p < 0.05). Extinction on days 4 and 5 are depicted as 3 min bins. Bar graph depicts R1. (N, Veh = 14, 3 mpk = 7, 10 mpk = 11, 30 mpk = 11).

Figure 5:. M₁ Potentiation Enhances Fear Extinction in a Model of PTSD.

(A) Schematic illustrating the SEFL model and experimental design. On day 1, mice underwent SEFL conditioning in Context A where they received 10 footshocks at random intervals over 1 hour. Control mice were placed in Context A for 1 hour. Days 2, 3, and 4 were performed in a novel Context B. On day 2, mice were conditioned with 2 mild footshocks in Context B. On day 3, SEFL-conditioned mice were administered vehicle (20% β-cyclodextrin) or 10 mpk VU0453595 i.p. 15 min before being placed back in Context B where they underwent a 15 min context extinction session. On day 4, mice were placed back in Context B for 3 min to assess context extinction consolidation. (B) Mice that received SEFL on day 1 froze significantly more on day 2 during acquisition and (C) on day 3. Bar graph depicts first 3 min in Context B on day 3. (Unpaired student’s t-test, ***p < 0.001). (D) Administration of 10 mpk VU0453595 had no effect on within-session extinction on day 3 (Two-way repeated-measures ANOVA: Effect of Drug: F_1,18 = 5.033, p = 0.440; Effect of time block: F_4,18 = 15.15, p < 0.0001; Interaction: F_4,18 = 0.782, p = 0.541) but enhanced consolidation of contextual fear extinction measured on day 4. (Unpaired student’s t-test, *p < 0.05. N, CTL = 8, SEFL/Veh = 10)