Distinct projections from the infralimbic cortex exert opposing effects in modulating anxiety and fear

Abstract

Anxiety-related disorders can be treated by cognitive therapies and transcranial magnetic stimulation, which involve the medial prefrontal cortex (mPFC). Subregions of the mPFC have been implicated in mediating different and even opposite roles in anxiety-related behaviors. However, precise causal targets of these top-down connections among diverse possibilities have not been established. Here, we show that the lateral septum (LS) and the central nucleus of the amygdala (CeA) represent 2 direct targets of the infralimbic cortex (IL), a subregion of the mPFC that modulates anxiety and fear. Two projections were unexpectedly found to exert opposite effects on the anxious state and learned freezing: the IL-LS projection promoted anxiety-related behaviors and fear-related freezing, whereas the IL-CeA projection exerted anxiolytic and fear-releasing effects for the same features. Furthermore, selective inhibition of corresponding circuit elements showed opposing behavioral effects compared with excitation. Notably, the IL-CeA projection implemented top-down control of the stress-induced high-anxiety state. These results suggest that distinct IL outputs exert opposite effects in modulating anxiety and fear and that modulating the excitability of these projections with distinct strategies may be beneficial for the treatment of anxiety disorders.

Keywords: Neurological disorders; Neuroscience.

Figure 1. The IL is implicated in anxiety-like behavior.

(A) Expression of ChR2 in the IL. Scale bar: 500 μm. M2,secondary motor cortex; Cg1, cingulate cortex, area 1; PL, prelimbic cortex. (B) Brief blue light pulses at 5 Hz, 20 Hz, and 40 Hz precisely activated ChR2 cells. (C) Schematic protocol for investigating the behavioral impact of optogenetic activation of IL somata. (D) Representative animal track across epochs in the EPM for a ChR2 mouse. Mice were tested in 3-minute epochs across a 9-minute session. (E) ChR2 mice (n = 10) spent less time in the open arms than did eYFP mice (n = 8) during photostimulation [F_interaction (2,32) = 3.959, P = 0.0291]. (F) ChR2 mice showed a lower probability of entering the open arms than did eYFP mice during photostimulation [F_interaction (2,32) = 10.88, P = 0.0002]. (G) Representative animal track across epochs in the OFT for a ChR2 mouse. (H) During the illumination epoch, ChR2 mice spent less time exploring the center of the open field than did eYFP mice [F_interaction (2,32) = 21.12, P < 0.0001]. (I) Photostimulation did not alter the distance traveled for mice in either group. (J) Expression of eNpHR in the IL. Scale bar: 500 μm. (K) Yellow light illumination of eNpHR cells in the IL blocked evoked spiking. (L) Schematic protocol for investigating the behavioral impact of optogenetic inhibition of IL somata. (M) Same as in E but for eNpHR mice [n = 7 per group, F_interaction (2,24) = 4.452, P = 0.0227]. (N) Same as in F but for eNpHR mice [F_interaction (2,24) = 7.726, P = 0.0026]. (O) Same as in H but for eNpHR mice [F_interaction (2,24) = 11.76, P = 0.0003]. (P) Same as in I but for eNpHR mice. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA with Bonferroni’s post hoc analysis (E–I and M–P). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 2. Distinct IL outputs modulate opposite anxiety-related behaviors.

(A and B) Confocal images of coronal sections showing IL terminals in the LS (A), BLA, and CeA (B). Scale bars: 200 μm. (C) Experimental design to investigate the behavioral impact of optogenetic activation of IL-LS or IL-CeA projections. (D) eYFP and ChR2 mice expressing eYFP or ChR2-eYFP, respectively, in the IL with optic fibers above the LS. n = 15 eYFP mic; n = 13 ChR2 mice. (E–H) Blue light decreased the exploration of open arms [E, F_interaction (2,52) = 8.447, P = 0.0007], as well as the probability of open-arm entry [F, F_interaction (2,52) = 11.35, P = 0.0002] and center exploration in the open field [G, F_interaction (2,52) = 11.90, P < 0.0001], without altering locomotion [H, F_interaction (2,52) = 0.1149, P = 0.8917]. (I–M) Same as in D–H, but for IL-CeA projections. n = 8 eYFP mice; n = 8 ChR2 mice. Yellow light increased the exploration of open arms [J, F_interaction (2,28) = 7.20, P = 0.0030], resulted in a higher probability of open-arm entry [K, F_interaction (2,28) = 7.264, P = 0.0029], increased center exploration in the open field [L, F_interaction (2,28) = 12.18, P = 0.0002], and did not alter locomotion [M, F_interaction (2,28) = 0.3525, P = 0.7060]. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA with Bonferroni’s post hoc analysis (E–H and J–M). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 3. Glutamatergic IL inputs into the LS or CeA modulate anxiety-related behaviors.

(A) Experimental paradigm. Experiments were performed 7 or 8 weeks after AAV2-CaMKIIα-ChR2-eYFP injection. Thirty minutes before the behavioral assays and laser stimulation, glutamate receptor antagonists (GluR antag: AP5 + NBQX) or saline were unilaterally infused locally into the LS or CeA using the same guide cannula that delivered light via an optical fiber. (B) Mice expressing ChR2 in the IL were treated with saline or AP5 plus NBQX via cannulas above the LS. (C–F) Relative to saline injections, AP5 plus NBQX injections into the LS blocked the light-induced decreases in open-arm exploration on the EPM test [C, F_interaction (2,44) = 12.57, P < 0.0001; D, F_interaction (2,44) = 17.71, P = 0.000] and center exploration in the open field [E, F_interaction (2,30) = 3.392, P = 0.0470]. Neither light stimulation nor injection of AP5 plus NBQX altered the total distance traveled [F, F_interaction (2,30) = 0.2107, P = 0.8112]. (G–K) Same as in B–F, but for IL-CeA projections. After intra-CeA glutamate receptor blockade, photoactivation of ChR2-expressing IL terminals in the CeA failed to increase open-arm exploration [H, F_interaction (2,38) = 3.100, P = 0.0566; I, F_interaction (2,38) = 13.84, P < 0.0001], center exploration in the open field [J, F_interaction (2,34) = 7.009, P = 0.0028], or locomotion [K, F_interaction (2,34) = 0.08985, P = 0.7863]. n = 11 and n = 13 mice for C and D; n = 9 and n = 8 mice for E and F; n = 11 and n = 10 mice for H and I; n = 10 and n = 9 mice for J and K. *P < 0.05 and ***P < 0.001., by 2-way, repeated-measures ANOVA with Bonferroni’s post hoc analysis (C–F and H–K). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 4. Monosynaptic and functional IL inputs into the LS or CeA.

(A and D) Experimental scheme. Two methods were used: injection of retrobeads into the LS (A) or CeA (D) and injection of CAV-Cre virus into the LS (A) or CeA (D) and AAV-DIO-ChR2-Cherry virus into the IL (A and D). (B) Fluorescence images illustrating retrobead back-labeled LS-projecting IL neurons and graph indicating the total number of these neurons. LV, lateral ventricle. (C) Same as in B, but for mCherry expression. (E) Same as in B, but for CeA-projecting IL neurons. (F) Same as in E, but for mCherry expression. (G and K) Scheme for recording postsynaptic currents in the LS (G) or CeA (K) evoked by optogenetic activation of IL projections. (H and L) Overlay of light-triggered responses in the LS (H) and CeA (L). (I and M) Light-evoked postsynaptic currents were completely blocked by TTX and recovered by TTX plus 4AP, which were blocked by 6-cyano-7-nitroquinoxaline-2,3-dioneis (CNQX) [I, n = 7 neurons from 2 mice, F(3,24) = 27.01, P < 0.0001; M, n = 5 neurons from 2 mice, F(3,16) = 26.54, P < 0.0001; 1-way ANOVA with Tukey’s multiple-comparison test]. (J and N) Onset latencies for the LS (J) and CeA (N). (O) Experimental scheme showing that CTB was injected into 2 targets of IL neurons, the LS and the CeA. (P) Fluorescence images illustrating CTB targeted to the LS (CTB488, green) and the CeA (CTB555, red). (Q) Coronal section of the IL labeled with CTB in green (LS) and red (CeA). (R) Number of IL neurons labeled with CTB. LS-projecting IL neurons, n = 519 ± 13 cells (average from 3 mice; 459 [green circle] + 60 [yellow overlap] = 519); CeA-projecting IL neurons, n = 228 ± 11 cells (average from 3 mice; 168 [red circle] + 60 [yellow overlap] = 228). Scale bars: 100 μm (B, C, E, F, and Q) and 500 μm (P). ***P < 0.001, by 1-way ANOVA with Tukey’s multiple-comparison test (I and M). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 5. IL-LS and IL-CeA inhibition shows opposite effects on anxiety-related behaviors.

(A) Experimental paradigm for optogenetic inhibition. Neurons in the IL were transduced with either NpHR-eYFP or eYFP. Yellow light was delivered via bilateral optical fibers implanted into the LS or CeA after 6–7 weeks of viral incubation. (B–D) eNpHR IL-LS mice, compared with eYFP mice, showed increased open-arm exploration [B, F_interaction (4,38) = 15.69, P < 0.0001] and a higher probability of open-arm entry [C, F_interaction (4,38) = 17.89, P < 0.0001] in the EPM test and increased center exploration time [D, F_interaction (4,38) = 18.38, P < 0.0001] in the OFT during the illumination epoch, whereas yellow light induced opposite effects on the NpHR-IL-CeA mice. n = 8 eYFP mice; n = 7 eNpHR-LS mice; n = 7 eNpHR-CeA mice. (E) total distance traveled. (F) Experimental paradigm for pharmacogenetic inhibition. Neurons in the IL were transduced with either hM4DGi-mCherry or mCherry. CNO was delivered via bilateral guide cannula implanted into the LS or CeA 30 minutes before the behavioral assays, after 6 to 7 weeks of viral incubation. (G–I) hM4DGi IL-LS mice, compared with mCherry mice, showed an increase in both open-arm exploration in the EPM test [G, F(2,25) = 36.92, P < 0.0001; H, F(2,25) = 32.10, P < 0.0001] and center exploration in the OFT [I, F(2,25) = 80.83, P < 0.0001] after CNO infusion, whereas CNO induced opposite effects on the hM4DGi IL-CeA mice. n = 9 eYFP mice; n = 10 eNpHR-LS mice; n = 9 eNpHR-CeA mice. (J) Total distance traveled. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA with Dunnett’s multiple-comparison test (B–E) and 1-way ANOVA with Dunnett’s multiple-comparison test (G–J). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 6. IL-LS and IL-CeA circuits modulate fear extinction in the opposite direction.

(A) Scheme of mice expressing ChR2 or eYFP in the IL with optic fibers above the LS or CeA. (B) Effects of optogenetic activation of IL-LS (ChR2-LS) and IL-CeA (ChR2-CeA) projections on fear retrieval, extinction, and extinction retrieval. Blue light was delivered on day 2 and day 3. Mice showed similar fear retrieval in each group [F(2,23) = 0.04646, P = 0.9547]. ChR2-LS mice froze more than did eYFP control mice, while ChR2-CeA mice froze less during fear extinction [F_interaction (18,207) = 3.54, P < 0.0001] and extinction retrieval [F(2,23) = 17.23, P < 0.0001]. n = 10 eYFP mice; n = 8 ChrR2-LS mice; n = 8 ChR2-CeA mice. (C) Scheme of mice expressing hM4DGi or mCherry in the IL with a cannula above the LS or CeA. (D) Effects of pharmacogenetic inhibition of IL-LS (hM4DGi-LS [Gi-LS]) and IL-CeA (hM4DGi-CeA [Gi-CeA]) projections on fear retrieval, extinction, and extinction retrieval. CNO was delivered 30 minutes before fear retrieval and extinction. Mice showed similar degrees of fear retrieval in each group [F(2,25) = 0.4312, P = 0.6545]. hM4DGi-LS mice froze less than did mCherry control mice, whereas hM4DGi-CeA mice froze more during fear extinction [F_interaction (18,230) = 3.336, P < 0.0001] and extinction retrieval [F(2,25) = 25.88, P < 0.0001]. n = 9 mCherry mice; n = 10 hM4DGi-LS mice; n = 9 mice hM4DGi-CeA mice. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Dunnett’s multiple-comparison test for fear retrieval and extinction retrieval and 2-way, repeated-measures ANOVA with Dunnett’s multiple-comparison test for fear extinction. Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 7. Activation of CeL- and CeM-projecting IL neurons differentially regulates fear-related behaviors.

(A, B, and D) Experimental paradigm. Injection of CAV-Cre virus into the CeM (B) or CeL (D) and injection of AAV-DIO-ChR2-mCherry virus into the IL. Blue light was delivered via optical fibers implanted into the IL during the behavioral tests. (C) Image of mCherry expression in CeM-projecting IL neurons and graph showing the total number of CeM-projecting IL neurons. Scale bar: 200 μm. (E) Same as in D, but for CeL-projecting IL neurons. Scale bar: 200 μm. (F–H) CeM-ChR2–projecting mice showed increased open-arm exploration [F, F_interaction (4,38) = 15.69, P < 0.0001] and a higher probability of open-arm entry [G, F_interaction (4,38) = 17.89, P < 0.0001] in the EPM test, as well as an increase in center exploration time [H, F_interaction (4,38) = 18.38, P < 0.0001] in the OFT during the illumination epoch, whereas blue light only induced an increase in center exploration time [H, F_interaction (4,38) = 18.38, P < 0.0001] in the OFT for CeL-ChR2–projecting mice. (I) Total distance traveled. (J) Effects of optogenetic activation of CeM-ChR2 and CeL-ChR2 mice on fear retrieval, extinction, and extinction retrieval. CeM-ChR2 mice froze less than did mCherry control mice, while CeL-ChR2 mice showed levels of freezing similar to those of mCherry control mice during fear extinction [F_interaction(2,20) = 13.51, P = 0.0002] and extinction retrieval [F(2,22) = 45.12, P < 0.0001]. n = 9 mCherry mice; n = 8 CeM-ChR2 mice; n = 8 CeL-ChR2 mice. *** P < 0.001, by 2-way, repeated-measures ANOVA with Dunnett’s multiple-comparison test (F–I) and 1-way ANOVA with Dunnett’s multiple-comparison test for fear retrieval and extinction retrieval and 2-way, repeated-measures ANOVA with Dunnett’s multiple-comparison test for fear extinction (J). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details.

Figure 8. IL-CeA projections reverse the anxiogenic effects of restraint stress.

(A) Experimental paradigm. Neurons in the IL were transduced with either ChR2 or eYFP. Blue light was delivered via optical fibers implanted into the CeA after 3 days of restraint stress. (B–E) Compared with control mice, mice that underwent restraint stress showed a reduction of both open-arm exploration in the EPM test [B, F_interaction (6,63) = 8.89, P < 0.0001]; [C, F_interaction (6,64) = 11.40, P < 0.0001] and center exploration in the OFT [D, F_interaction (6,64) = 16.20, P < 0.0001]. IL-CeA activation reversed these stress-induced effects. No detectable effect was seen on the distance traveled by mice in each group [E, F_interaction (6,64) = 0.1177, P = 0.9940]. n = 9 control-eYFP mice; n = 9 stress-eYFP mice; n = 9 control-ChR2 mice; n = 8 stress-ChR2 mice. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA with Tukey’s multiple-comparison test (B–E). Data are presented as the mean ± SEM. See Supplemental Table 1 for statistical details. Con, control.