The NRG1-ErbB4 signaling pathway in the ITC area is required for fear extinction and inhibition in CeM neurons.
GABAergic ITC neurons are critical for the modulation of fear memory extinction. Although we observed that ErbB4 was found primarily in GABAergic interneurons in the BLA [24], there is no morphological or behavioral evidence to show whether ErbB4 is expressed in GABAergic ITC neurons or is involved in the regulation of fear extinction. To address this issue, we first explored whether ErbB4 is expressed primarily in the interneurons of the ITC area using transgenic mice to identify the colocalization of GAD67-positive interneurons and ErbB4-positive neurons. Figure 1A shows the coimmunofluorescence image of tdTomato (red) reporting ErbB4 expression and GAD67 expression (green) in the ITC. Most ErbB4-expressing neurons in the ITC area coexpressed GAD67 (98.99±0.007%; Figure 1A). These results provide histological evidence that ErbB4 is abundantly expressed in GABAergic ITC neurons.
Based on previous studies suggesting important roles for the ITC area in fear extinction and the NRG1-ErbB4 pathway in GABAergic neurotransmission, we hypothesized that the ITC area was the amygdalar subregion requiring the NRG1-ErbB4 pathway in fear extinction. To test this hypothesis, the drug was infused into the ITC area. One hour later, mice underwent fear extinction training. We observed significantly reduced freezing levels during fear extinction in the NRG1 group than in the control group (F3, 684 = 71.943, P < 0.001; Figure 1C). Mice treated with ErbB4’s antagonist (AG1478) exhibited impaired fear extinction compared with the control mice (F3, 684 = 71.943, P < 0.001; Figure 1C). There were no significant differences between the AG1478 group and NRG1+AG1478 group (F3, 684 = 71.943, P = 0.56; Figure 1C). Taken together, these findings suggest that NRG1 promotes fear extinction through ErbB4 receptors in the ITC area.
To test whether extinction depends on NRG1-mediated increased feed-forward inhibition in the CeM, we first compared responses of CeM neurons to BLA inputs (Figure 1D) in amygdala slices obtained from mice that were previously treated with AAV-ectoErbB4 and subjected to fear extinction versus control mice that were treated with AAV-GFP and trained on extinction. AAV-ectoErbB4 was designed to express a polypeptide that includes the entire extracellular domain of ErbB4 and binds to NRG1, preventing NRG1 from activating endogenous receptors [34]. We confirmed that expression of the virus was limited only in the ITC three weeks after virus injection (Supplementary Figure 1). We also identified ITC neurons projected to the CeM (Supplementary Figure 2).
We examined the behavior of these mice (Figure 1F) and then analyzed the responsiveness of CeM neurons to BLA inputs in vitro (Figure 1G, 1H). Analysis of percent time spent freezing (Figure 1F) demonstrated that the Ecto-E group exhibited higher levels of conditioned freezing during fear extinction (day 5) than the control group (F2, 513 = 392.994, P < 0.001; Figure 1F). These observations thus confirm the conclusion that NRG1 in the ITC area is necessary for the regulation of fear extinction.
We anesthetized the mice 24 h after exposure to the extinction training context and prepared coronal sections from their amygdala tissues. We obtained patch recordings from samples of 10 CeM neurons per group and compared their responsiveness to electrical stimuli delivered at a standard position in the BLA (Figure 1D). The eIPSC amplitudes of CeM neurons from the Ecto-E mice and the Control-No E mice were significantly lower than those from the Control-E mice (F2, 27 = 5.113, P =0.007 and 0.016, respectively; Figure 1G and 1H). However, eIPSCs from Ecto-E mice were not significantly different from those of the Control-No E group (F2, 27 = 5.113, P =0.719; Figure 1G and 1H). Blocking ITC NRG1 did not affect the increased excitatory input to CeM neurons by stimulating BLA neurons (Supplementary Figure 3). Next, we investigated the presynaptic or postsynaptic mechanism involved in the regulation of GABAergic transmission by NRG1. The mIPSCs were measured at CeM neurons. The mIPSC frequencies, but not the mIPSC amplitudes, of the Control-E group were higher than those of the Control-No E group (F2, 27 = 6.203 and 0.197, P =0.005 and 0.619, respectively; Figure 1I-1K). Inhibiting NRG1 produced no effect on the mIPSC amplitudes but significantly decreased the frequencies compared to the Control-E group (F2, 27 = 0.197 and 6.203, P =0.941 and 0.005, respectively; Figure 1I-1K). These results indicate that fear extinction is associated with enhanced BLA-evoked IPSCs in the CeM that could be reversed by inhibiting ITC NRG1. ITC NRG1 is important for the regulation of BLA-evoked presynaptic inhibition of the CeM, which is involved in fear extinction.
Blocking ITC NRG1 does not affect the increased excitatory input to ITC neurons from BLA neurons but leads to decreased inhibition of CeM neurons.
To explore how extinction depends on increased inhibition of the CeM mediated by ITC NRG1, we first compared the responses of ITC neurons to BLA inputs (Figure 2A) in amygdala slices obtained from mice treated with AAV-ectoErbB4 and subjected to fear extinction versus control mice treated with AAV-GFP and trained on extinction (Figure 1E).
We anesthetized the mice 24 h after extinction training and prepared amygdala slices. We first obtained patch recordings of the ITC neurons and compared their responsiveness to the electrical stimuli delivered at the BLA (Figure 2A). Thereafter, we measured evoked excitatory postsynaptic currents (eEPSCs) in ITC neurons. AP5 (100 mM) and CNQX (20 mM) were included in ACSF when we recorded AMPAR-EPSCs and NMDAR-EPSCs, respectively. The AMPAR-EPSC amplitudes of ITC neurons from the Ecto-E mice and the Control-E mice were significantly higher than those from the Control-no E mice (F2, 27 = 6.356, P =0.019 and 0.009, respectively; Figure 2B). However, AMPAR-EPSCs from Ecto-E mice were not significantly different from those of the Control-E group (F2, 27 = 6.356, P =0.993; Figure 2B). However, the NMDAR-EPSCs showed no differences between these groups (F2, 27 = 0.097, P =0.908; Figure 2D).
Next, we investigated the responses of CeM neurons to ITC inputs (Figure 2F) in amygdala slices obtained from mice treated with AAV-ectoErbB4 and subjected to fear extinction versus control mice that were treated with AAV-GFP and trained on extinction. We then obtained patch recordings of the CeM neurons and compared their responsiveness to the electrical stimuli delivered at the ITC (Figure 2A). The eIPSC amplitudes of CeM neurons from the Control-E mice were significantly larger than those from the Control-no E mice (F2, 27 = 5.147, P =0.028; Figure 2H). However, the eIPSCs from Ecto-E mice were significantly smaller than those of the Control-E group (F2, 27 = 5.147, P =0.031; Figure 2H). These results indicate that fear extinction is associated with enhanced BLA-evoked EPSCs in the ITC, which elicits feed-forward inhibition of CeM neurons. The feed-forward inhibition of CeM elicited by ITC neurons is regulated by the NRG1 signaling pathway.
ITC NRG1 might regulate GABAergic transmission through P/Q-type VACCs but not through L- or N-type VACCs.
The decreased IPSCs observed after blocking NRG1 were mediated by different neurotransmission mechanisms. One possibility is that they decrease Ca2+-dependent synaptic transmission. To examine this, we studied the function of an ErbB4 antagonist (AG1478) and inhibitors of VACC subtypes on eIPSC amplitudes of CeM neurons. We measured the eIPSCs of CeM neurons and compared their responsiveness to electrical stimuli delivered at the ITC (Figure 3A). A series of experiments were performed by additively applying AG1478 (5 μM) and one of the following blockers: L-type VACC blocker nifedipine (NFDP, 4 μM), N-type VACC blocker ω-Ctx-GVIA (CTx, 1 μM), or P/Q-type VACC blocker ω-Aga-IVA (Aga, 0.5 μM).
As shown in Figure 3B-3D, NFDP exerted weak blocking effects on eIPSCs on CeM neurons (<6%), whereas CTx and Aga had significant blocking effects on CeM neurons. Interestingly, by applying AG1478 after VACC antagonist treatments, we obtained significantly increased inhibition of the eIPSCs on NFDP- and CTx-treated neurons but not on Aga-treated pyramidal neurons [t(10) = -4.162, -3.925, and -0.665; P =0.002, 0.003 and 0.521, respectively; Figure 3B-3D], suggesting that the NRG1-ErbB4 signaling pathway may act through P/Q-type channels but not through L- or N-type channels. To further identify the possible effects of Ca2+ channel antagonists on the reaction of AG1478 in Figure 3, we analyzed the contribution of AG1478 in the absence or presence of Ca2+ channel antagonists. The decreased percentage of eIPSC amplitudes mediated by AG1478 did not change in the presence of L- or N-type channel antagonists (F3, 20 = 6.581, P =0.818, 0.851, respectively; Figure 3E). However, AG1478 barely worked in the presence of the P/Q-type antagonist Aga compared to the control group (F3, 20 = 6.581, P <0.001, Figure 3E). These data indicate that the NRG1-ErbB4 signaling pathway may regulate GABAergic transmission through P/Q-type channels but not through L- or N-type channels.
Since the above results suggest that the NRG1-ErbB4 signaling pathway might affect IPSCs via presynaptic mechanisms, we next directly measured the effects of NRG1-ErbB4 on VACCs of ITC interneurons (Figure 3F). Then, we additively applied the AG1478, Aga, CTx, and NFDP to measure VACC current changes. We found that AG1478 significantly decreased the amplitude of VACC currents (F4, 25 = 203.744, P =0.007, Figure 3G). We did not observe a significant reduction in VACC currents in response to Aga compared to the AG1478 treatment (F4, 25 = 203.744, P =0.895, Figure 3G). However, we found that VACC currents were significantly reduced by CTx in the presence of AG1478 and Aga (F4, 25 = 203.744, P =0.002, Figure 3G). NFDP further reduced VACC currents in the presence of AG1478, Aga and CTx (F4, 25 = 203.744, P =0.001, Figure 3G). To exclude the possible effect of AG1478 on Aga, we added Aga first, and AG1478 still did not cause a significant difference between the Aga group and the AG1478 group (F4, 25 = 98.538, P =0.295, Figure 3H). These data were quite coincident with the above conjecture that the NRG1-ErbB4 signaling pathway regulates GABAergic transmission through P/Q-type VACCs but not through L- or N-type VACCs.
Blocking ITC NRG1 has no effect on the excitability of BLA synapses projecting to the ITC but leads to decreased feed-forward inhibition of CeM neurons mediated by reduced P/Q-type VACC currents of ITC neurons.
To further support the function of ITC NRG1 in fear extinction and the underlying mechanism, we first compared the responses of ITC neurons to BLA inputs (Figure 4A) and then compared the responses of CeM neurons to BLA inputs (Figure 4D) in amygdala slices obtained from mice that were treated with AAV-ectoErbB4 and subjected to fear extinction versus control mice that were treated with AAV-GFP and trained on extinction. BLA glutamatergic projection neurons were transfected with adeno-associated virus serotype 5 (AAV5) carrying codon-optimized channel rhodopsin (ChR2)–egfp under the control of the CaMKIIa promoter. For all groups, the virus was injected into the ITC areas three weeks before the behavioral experiments. We anesthetized the mice 24 h after extinction training and prepared amygdala sections. We also confirmed that expression of the virus was limited only in the BLA and identified that BLA neurons projected to the ITC three weeks after virus injection (Supplementary Figure 4).
We first obtained patch recordings from ITC neurons and compared their responsiveness to photostimulation of BLA terminals in the ITC (Figure 4A). We performed these tests at a membrane potential of −70 mV. The eEPSC amplitudes of ITC neurons from the Control-E mice and the Ecto-E mice were significantly higher than those from the Control-No E mice (F2, 27 = 5.091, P =0.042 and 0.023, respectively; Figure 4B and C). However, eEPSCs from Ecto-E mice were not significantly different from those of the Control- E group (F2, 27 = 5.091, P =1; Figure 4B and C). Next, we investigated the responses of CeM neurons to photostimulation of BLA terminals in the ITC (Figure 4D). The eIPSC amplitudes of CeM neurons from the Control-E mice were significantly higher than those from the Control-No E and Ecto-E mice (F2, 27 = 10.097, P =0.009 and 0.017, respectively; Figure 4E and 4F). However, eIPSCs from Ecto-E mice were not significantly different from those of the Control-No E group (F2, 27 = 10.097, P =1; Figure 4E and 4F). The latency of eIPSCs recorded on CeM neurons was longer than that of eEPSCs recorded on ITC neurons [t(58) =–8.766; P <0.001, Figure 4G]. Altogether, the above results again demonstrated that BLA glutamatergic neurons projected to ITC GABAergic neurons and elicited feed-forward inhibition of CeM neurons.
We next directly measured the effects of NRG1-ErbB4 on P/Q-type VACC currents of ITC interneurons and compared the amplitudes of P/Q-type VACC currents in coronal slices of the amygdala obtained from mice that were previously treated with AAV-ectoErbB4 and subjected to fear extinction versus control mice that were treated with AAV-GFP and trained on extinction (Figure 4I and 4J). We applied the P/Q-type VACC blocker Aga to measure the P/Q-type VACC current changes using Ba2+ as the charge carrier. We found that the amplitudes of P/Q-type VACC currents in the Control-E group were higher than those in the Control-No E group and in the Ecto-E group (F2, 15 = 7.619, P =0.005 and 0.011, respectively; Figure 4I and 4J). No differences were observed between the Control-No E group and the Ecto-E group (F2, 15 = 7.619, P =1; Figure 4I and 4J). These data again supported the above conjecture that the ITC NRG1-ErbB4 signaling pathway regulates GABAergic transmission through P/Q-type channels involved in fear extinction.
The ITC NRG1 signaling pathway might regulate fear extinction through P/Q-type VACCs.
These results indicate that the ITC NRG1-ErbB4 signaling pathway regulates BLA-evoked presynaptic inhibition of the CeM through P/Q-type VACC channels, which are involved in extinction training. To further test this, drug (control ACSF, NRG1, NRG1+Aga, Aga) was infused into the ITC area through guide cannulas, and 1 h later, mice underwent fear extinction training (Figure 5B). We observed significantly lower freezing levels during fear extinction in the NRG1 group than in the control group (F3, 513 = 1.706, P < 0.001; Figure 5C). Mice treated with NRG1+Aga or Aga exhibited impaired fear extinction compared to control mice (F3, 513 = 1.706, both P < 0.001; Figure 5C). No differences were observed between the NRG1+Aga and Aga groups (F3, 513 = 1.706, P =0.988; Figure 5C). These observations suggest that NRG1 promotes fear extinction through P/Q-type VACC channels in the ITC area.
To further elucidate the function of ITC NRG1 in fear extinction and the underlying mechanism, we compared the responses of CeM neurons to BLA inputs in amygdala slices (Figure 2; Figure 5A). We anesthetized mice 24 h after extinction training and prepared amygdala slices. We then measured the responses of CeM neurons to photostimulation of BLA terminals in the ITC (Figure 2; Figure 5D). The eIPSC amplitudes of CeM neurons from the NRG1-treated mice were significantly higher than those from the Control mice (F3, 37 = 17.726, P=0.044, Figure 5D and 5E). However, the amplitudes of eIPSCs from the NRG1+Aga group or the Aga group were significantly lower than those of the Control group (F3, 37 = 17.726, P =0.019 and 0.006, respectively; Figure 5D and 5E). No differences were observed between the NRG1+Aga group and the Aga group (F3, 37 = 17.726, P=0.728; Figure 5D and 5E). Altogether, the above results again support that BLA glutamatergic neurons elicited ITC feed-forward inhibition of CeM neurons during the fear extinction process, which could be regulated by ITC NRG1-ErbB4 signaling. The ITC NRG1-ErbB4 signaling pathway might regulate GABAergic neurotransmission required for fear extinction through P/Q-type channels.