Stress-induced changes of the cholinergic circuitry promote retrieval-based generalization of aversive memories

Abstract

Generalization, the process of applying knowledge acquired in one context to other contexts, often drives the expression of similar behaviors in related situations. At the cellular level, generalization is thought to depend on the activity of overlapping neurons that represent shared features between contexts (general representations). Using contextual fear conditioning in mice, we demonstrate that generalization can also occur in response to stress and result from reactivation of specific, rather than general context representations. We found that generalization emerges during memory retrieval, along with stress-induced abnormalities of septohippocampal oscillatory activity and acetylcholine release, which are typically found in negative affective states. In hippocampal neurons that represent aversive memories and drive generalization, cholinergic septohippocampal afferents contributed to a unique reactivation pattern of cFos, Npas4, and repressor element-1 silencing transcription factor (REST). Together, these findings suggest that generalization can be triggered by perceptually dissimilar but valence-congruent memories of specific aversive experiences. Through promoting the reactivation of such memories and their interference with ongoing behavior, abnormal cholinergic signaling could underlie maladaptive cognitive and behavioral generalization linked to negative affective states.

Fig 1:. SD triggers abnormalities of hippocampal cholinergic signaling contributing to SIG

A Top, experimental design of LFP recordings performed during pre- and post-stress retrieval tests. Middle, traces of LFP recordings in Contexts A and B before and after SD. Bottom, stress- and context-dependent changes of DH oscillatory activity, n = 5 mice. Bottom Left: Two-way RM ANOVA. Factor: Stress, F (1, 4) = 0.6211, p=0.4747. Factor: Context, F (1, 4) = 4.589, p=0.0988. Factor: Stress x Context, F (1, 4) = 1.159, p=0.3424. Bottom Center: Two-way RM ANOVA. Factor: Stress, F (1, 4) = 0.7193, p=0.4442. Factor: Context, F (1, 4) = 53.55, p=0.0019. Factor: Stress x Context, F (1, 4) = F (1, 4) = 47.50, p=0.0023. Bottom Right: Two-way RM ANOVA. Factor: Stress, F (1, 4) = 0.1706, p=0.7008. Factor: Context, F (1, 4) = 19.37, p=0.0117. Factor: Stress x Context, F (1, 4) = 0.5711, p=0.4919. B Top, experimental design of fiber photometry of ACh release in NS and SD groups in the home cage after exposure to Contexts A and B. Middle, dynamics of ACh release during context exposure in NS and SD mice. In NS mice, two-way RM ANOVA revealed a significant Time x Context interaction indicative of fluctuations of Ach levels during context tests {Factor: Time, F (7.226, 115.6) = 1.636, p=0.1297. Factor: Context, F (3, 16) = 0.3821, p=0.7673. Factor: Time x Context, F (96, 512) = 1.621, p=0.0005. n=5 mice per group]. Such fluctuations of ACh release were disrupted during context exposure in SD mice [Two-way RM ANOVA. Factor: Time, F (5.374, 53.74) = 1.156, p=0.3434. Factor: Context, F (1, 10) = 0.1402, p=0.7159. Factor: Time x Context, F 65, 650) = 0.7276, p=0.9456. n=6 mice per group]. Bottom, left, significant decrease of maximal amplitude during Context A tests of SD relative to NS mice [F(1,40 )= 6.127, p<0.0176 ]. Reduction of Ach fluctuations in SD mice was also found [F(1,40 )= 6.332, p<0.016], however, there were no significant post-hoc differences. C Top, experimental design of hippocampal muscarinic receptor inhibition with scopolamine. Cannulation was performed 72 hours before behavioral tests. Middle, lack of scopolamine effects on freezing behavior in NS mice. Middle left: Two-way RM ANOVA. Factor: Drug, F (1, 8) = 0.7626, p=0.4080. Factor: Context, F (1, 8) = 52.64, p<0.0001. Factor: Drug x Context, F (1, 8) = 0.1269, p=0.7309, n = 5 mice per group. Middle right: Mann Whitney Test. p=0.8810. Sum of Ranks, 28.5, 26.5 (Veh, Scop). Bottom, reduced generalization of freezing in SD mice injected with scopolamine. Bottom left: Two-way RM ANOVA. Factor: Drug, F (1, 16) = 14.86, p=0.0014. Factor: Context, F (1, 16) = 52.74, p<0.0001. Factor: Drug x Context, F (1, 16) = 7.576, p=0.0142. n = 9 mice per group. Bottom right: Mann Whitney Test. p=0.0014. Sum of Ranks, 119.5, 51.5 (Veh, Scop).

Fig 2:. SD triggers changes in MS oscillations and induces SIG through MS-DH projections

A Top, experimental design of LFP recordings performed during the pre- and post-stress retrieval tests. Middle, stress- and context-dependent changes of MS oscillatory activity. n=5 mice in each group. Middle Left: Two-Way RM ANOVA. Factor: Stress, F (1, 4) = 2.824, p=0.1682. Factor: Context, F (1, 4) = 19.65. p=0.0114. Factor: Context x Stress, F (1, 4) = 1.586. p=0.2764. Middle Center: Two-Way RM ANOVA. Factor: Stress, F (1, 4) = 13.93, p=0.0203. Factor: Context, F (1, 4) = 13.76. p=0.0207. Factor: Context x Stress, F (1, 4) = 4.104. p=0.1127. Middle Right: Two-Way RM ANOVA. Factor: Stress, F (1, 4) = 32.04, p=0.0048. Factor: Context, F (1, 4) = 62.88. p=0.0014. Factor: Context x Stress, F (1, 4) = 0.07422. p=0.7988, n=5 mice per group. Bottom, lack of changes in the directional coherence of MS and DH oscillatory activity. Bottom Left: Two-Way RM ANOVA. Factor: Stress, F (1, 4) = 0.3707, p=0.5755. Factor: Context, F (1, 4) = 0.1461. p=0.7218. Factor: Context x Stress, F (1, 4) = 0.6957. p=0.4512. Bottom Center: Two-Way RM ANOVA. Factor: Stress, F (1, 4) = 0.1235, p=0.7430. Factor: Context, F (1, 4) = 0.07882. p=0.7928. Factor: Context x Stress, F (1, 4) = 0.3309. p=0.5959. Bottom Right: Two-Way RM ANOVA. Factor: Stress, F (1, 4) = 0.7412, p=0.4378. Factor: Context, F (1, 4) = 0.008319. p=0.9317. Factor: Context x Stress, F (1, 4) = 0.09754. p=0.7704. B Visualization of MS neurons and MS to DH projections after injection of Cre-dependent AAV-GFP in Chat-Cre mice. C Top, localization of inhibitory DREADD and CNO infusions used for inhibition of MS to DH cholinergic projections by combining AAV-hM4Gi expression in MS to DH projections with injection of CNO in the DH. Cannulation was performed 72 hours before behavioral tests. Middle, experimental design depicting the timing of different circuit and behavioral manipulations. Bottom, reduced generalization of freezing in SD mice injected into DH with CNO. Bottom Left: Two-way RM ANOVA. Factor: Drug, F (1, 12) = 10.41, p=0.0073. Factor: Context, F (1, 12) = 20.03, p=0.0008. Factor: Drug x Context, F (1, 12) = 8.858, p=0.0116. n = 6 mice in Veh group, n = 8 mice in CNO group. Bottom Right: Mann Whitney Test. Sum of Ranks: 66 (Veh), 39 (CNO). p=0.0043.

Fig 3:. Optogenetic stimulation of MS-DH projections increases freezing in Context B.

A Timeline of opsin infusion in the MS and optogenetic stimulation of the DH. B Tonic 4 Hz optogenetic stimulation of DH did not affect freezing during the retrieval test in context B. The stimulation schedule is shown on the left, individual freezing responses during alternating on- or off-stimulation cycles are shown in the middle, and the combined data are on the right. Paired T-test, p=0.7445. n=10 mice. C Tonic 20 Hz optogenetic stimulation of DH did not affect freezing during the retrieval test in Context B. The stimulation schedule is shown on the left, individual freezing responses during alternating on- or off-stimulation cycles are shown in the middle, and the combined data are on the right. Paired T-test, p=0.8769. n=10 mice. D Burst 20 Hz optogenetic stimulation of DH significantly increased freezing in Context B in mice trained in Context A. The stimulation schedule is shown on the left, individual freezing responses during alternating on- or off-stimulation cycles are shown in the middle, and the combined data are on the right. Paired T-test, p=0.0022. n=21 mice. E A similar effect of burst 20 Hz optogenetic stimulation of DH resulting in generalization of freezing is found using a between subject design. Two-way ANOVA. Factor: Device, F (1, 18) = 1.244, P=0.2793. Factor: Context, F (1, 18) = 16.34, P=0.0008. Factor: Device x Context, F (1, 18) = 1.287, P=0.2714. n = 5 mice in each dummy group (A group, B group), n = 6 mice in each device group (A group, B group).

Fig 4:. SD alters the reactivation of NRAM-GFP expressing neurons assessed by co-localization with cFos and Npas4

A Left, timeline of AAV9-N-RAM-d2tTA-TRE-GFP infusion, Dox regimen, and retrieval tests. As in the previous experiment, labeling of DH neurons with NRAM-GFP was performed at the first retrieval test in Context A, whereas reactivation was assessed after the second retrieval test either in Contexts A (A-A) or B (B-B). Middle, illustration of triple labeling of DG neurons with GFP, cFos, and Npas4. Right, illustration of GFP labeling in mice kept ON-Dox during the entire behavioral paradigm. B The number of GFP-, cFos-, and Npas4-positive DG neurons and their combinations after the post-stress retrieval test of SD mice or corresponding retrieval test of NS mice. The number of Npas4/GFP-co-labelled neurons was significantly increased after retrieval in Context B, whereas the number of triple labeled cFos/Npas4/GFP neurons was only detected in SD but not NS mice. *p < 0.05. Two-Way ANOVA. GFP: Stress F (1, 24) = 1.127, P=0.2990. Context F (1, 24) = 0.0001643, P=0.9899. Stress x Context F (1, 24) = 0.3764, P=0.5453. n = 6–8 mice in each group. GFP-cFos: Stress F (1, 24) = 0.1462, P=0.7056. Context F (1, 24) = 0.4782, P=0.4959. Stress x Context F (1, 24) = 0.6292, P=0.4354. n = 6–8 mice in each group. GFP-Npas4: Stress F (1, 24) = 0.0362, P=0.8506. Context F (1, 24) = 2.147, P=0.1558. Stress x Context F (1, 24) = 0.08050, P=0.7791. n = 6–8 mice in each group. cFos: Stress F (1, 24) = 2.324, P=0.1404. Context F (1, 24) = 0.05052, P=0.8241. Stress x Context F (1, 24) = 0.8190, P=0.3745. n = 6–8 mice in each group. Npas4: Stress F (1, 24) = 0.08408, P=0.7743. Context F (1, 24) = 1.762, P=0.1968. Stress x Context F (1, 24) = 0.6845, P=0.4162. n = 6–8 mice in each group. cFos-Npas4: Stress F (1, 24) = 0.05915, P=0.8099. Context F (1, 24) = 1.111, P=0.3024. Stress x Context F (1, 24) = 0.4367, P=0.5024. n = 6–8 mice in each group. cFos-GFP/GFP: Stress F (1, 25) = 0.01053, P=0.9191. Context F (1, 25) = 2.917, P=0.1000. Stress x Context F (1, 25) = 0.5415, P=0.4687. n = 6–8 mice in each group. Npas4-GFP/GFP: Stress F (1, 25) = 0.02197, P=0.8834. Context F (1, 25) = 4.470, P=0.0446. Stress x Context F (1, 25) = 0.04054, P=0.8421. n = 6–8 mice in each group. cFos-Npas4-GFP/GFP: Stress F (1, 24) = 8.682, P=0.0070. Context F (1, 24) = 8.682, P=0.0070. Stress x Context F (1, 24) = 8.682, P=0.0070, n = 6–8 mice in each group. C In the A-A condition, all reactivated GFP neurons were positive for cFos, whereas in the A-B condition 18–20% of the reactivated neurons were Npas4-positive. Depending on the stress condition, the Npas4/GFP neurons were either a separate neuronal population (NS group) or a population co-labeled with cFos (SD).

Fig 5:. Chemogenetic inhibition of NRAM- but not FRAM-expressing neurons alleviates SIG

A Left, expression of FRAM-hM4Gi (AAV9-TRE-hM4Di-mCherry and AAV9-F-RAM-d2tTA-sEF1α-GFP) in DG. Right, timeline of the experimental manipulations. Cannulation was performed 72 hours before behavioral tests. B Left, freezing before or after SD in mice injected with Veh or CNO. SD significantly increased generalization. CNO did not affect freezing during the pre- or post-stress test. Right, similar effects were found with analysis of the generalization index. Left: Three-Way RM ANOVA. Factor: Drug, F (1, 17) = 0.7190, P=0.4083. Factor: Stress, F (1, 17) = 10.09, P=0.0055. Factor: Context, F (1, 17) = 55.95. P<0.0001. Factor: Context x Stress, F (1, 17) = 7.235. P=0.0155. Factor: Drug x Stress, F (1, 17) = 0.5723, P=0.4597. Factor: Drug x Context, F (1, 17) = 0.002591, P=0.9600. Factor: Stress x Context, F (1, 17) = 7.235, P=0.0155. Factor: Drug x Stress x Context, F (1, 17) = 0.0003833, P=0.9846. n=9–10 mice per group. Right: Two-Way RM ANOVA. Factor: Stress, F (1,17) = 9.367, p=0.0071. Factor: Drug, F (1,17) = 0.0005083, p=0.9251. Factor: Stress x Drug, F (1,17) = 0.01760, p=0.8960. n=10 mice per group. C Left, expression of NRAM-hM4Gi (AAV9-TRE-hM4Di-mCherry and either AAV9-N-RAM-d2tTA-sEF1α-GFP) in DG. Right, timeline of the experimental manipulations. Cannulation was performed 72 hours before behavioral tests. D Left, freezing before or after SD in mice injected with Veh or CNO. SD significantly increased generalization, an effect significantly reduced by CNO. Right, SD significantly increased the generalization index, whereas CNO significantly attenuated this effect. Left: Three-Way RM ANOVA. Factor: Drug, F (1, 18) = 6.278, P=0.0221. Factor: Stress, F (1, 18) = 8.571, P=0.0090. Factor: Context, F (1, 18) = 149.7. P<0.0001. Factor: Context x Stress, F (1, 18) = 17.14. P=0.0006. Factor: Drug x Stress, F (1, 18) = 2.660, P=0.1203. Factor: Drug x Context, F (1, 18) = 2.833, P=0.1096. Factor: Stress x Context, F (1, 18) = 17.14, P=0.0006. Factor: Drug x Stress x Context, F (1, 18) =1.993, P=0.1751. n=10 mice per group. Right: Two-Way RM ANOVA. Factor: Stress, F (1,18) = 8.912, p=0.0079. Factor: Drug, F (1,18) = 5.823, p=0.0267. Factor: Stress x Drug, F (1,18) = 2.135, p=0.1612. n=10 mice per group.

Fig 6:. SD-induced reduction of REST levels in NRAM-GFP neurons was reversed by chemogenetic inhibition of MS-DH cholinergic projections

A Left, illustration of co-labelling of REST (red) and NRAM-GFP (green) in the DG. Middle, illustration of nuclear localization of REST in NS mice, and its reduction in SD mice. Right, analysis was performed and quantified for REST/GFP-positive neurons, showing a significant reduction of nuclear REST signals in this population. Two-Way ANOVA. Factor: Stress, F (1, 155) = 64.12, p<0.0001. Factor: Context, F (1, 155) = 0.1531, p=0.6961. Factor: Stress x Context, F (1, 155) = 1.381, p=0.2418. n= 35–43 per group. B Illustration of REST/NRAM-GFP co-labeling in NS and SD mice after exposure to Contexts A or B. C Left, localization of AAV-DIO hM4Gi infusion into MS, NRAM-GFP into DH, and cannula implantation in the DH. Right, timeline of the infusions, implantation, Dox regimen, and behavioral manipulations. D Left, injection of CNO partially but significantly restored REST levels. Dashed line corresponds to the mean density of REST signal in the NS group. Student’s T Test, t=2.742, df=45, p=0.0087, n=23–24 per group. Right, REST immunolabeling in the DG of SD mice counterstained with Hoechst (top) or co-labeled with NRAM-GFP (bottom). E Left, in NRAM-GFP labeled neurons, CNO decreased the levels of Npas4 levels. Dashed line corresponds to the number of Npas4/GFP-positive cells after exposure to context A. Student’s T Test, t=3.798, df=12, p=0.0025, n=7 mice in each group. Right, illustration of immunolabeling of the DG (top) and individual neuronal populations (bottom).