Enhanced harm detection following maternal separation: Transgenerational transmission and reversibility by inhaled amiloride

Abstract

Early-life adversities are associated with altered defensive responses. Here, we demonstrate that the repeated cross-fostering (RCF) paradigm of early maternal separation is associated with enhancements of distinct homeostatic reactions: hyperventilation in response to hypercapnia and nociceptive sensitivity, among the first generation of RCF-exposed animals, as well as among two successive generations of their normally reared offspring, through matrilineal transmission. Parallel enhancements of acid-sensing ion channel 1 (ASIC1), ASIC2, and ASIC3 messenger RNA transcripts were detected transgenerationally in central neurons, in the medulla oblongata, and in periaqueductal gray matter of RCF-lineage animals. A single, nebulized dose of the ASIC-antagonist amiloride renormalized respiratory and nociceptive responsiveness across the entire RCF lineage. These findings reveal how, following an early-life adversity, a biological memory reducible to a molecular sensor unfolds, shaping adaptation mechanisms over three generations. Our findings are entwined with multiple correlates of human anxiety and pain conditions and suggest nebulized amiloride as a therapeutic avenue.

Fig. 1.. RCF is associated with altered homeostatic responses trigenerationally (data are represented as means + SEM).

(A) Association (n = 77) between 6% CO₂-air breathing and higher Vt compared to air breathing (Hotelling’s trace F_1,65 = 159.0, P ≤ 0.0001), with interaction between lineage (RCF/CT) and Vt during 6% CO₂ breathing (Hotelling’s trace, F_1,65 = 21.82, P ≤ 0.0001) by repeated-measures analyses of variance (ANOVA-R). There were no other significant associations owing to generation (F0, F1, and F2), sex, or any possible combinations of the predictors. (B) Association (n = 102) between shorter latency of paw withdrawal at thermal stimulation (Hargreaves’ test) and the RCF lineage (F₁ =199.70, P ≤ 0.001) by univariate GLM (F₁₁ = 22.36, P ≤ 0.001). There was also an association with generation (F0, F1, and F2: F₂ = 8.09, P ≤ 0.001): Tests of within- and between-groups’ variance decomposition showed that the latter, generation effect was caused by a local increase in paw withdrawal latency among F1-CT animals and revealed no lineage-by-generation interaction and no significant effect in multiple comparisons of between-generations’ effect (by Scheffe’s tests). No other significant associations owing to sex or to interactions among any of the independent variables emerged, including lineage-by-generation interaction. (C) Association (n = 81) between shorter latency of paw withdrawal at mechanosensitive stimulation [simplified up-down (SUDO)–von Frey’s test] and the RCF lineage (F₁ = 62.26, P ≤ 0.001) by univariate GLM (F₁₁ = 7.99, P ≤ 0.001). *P < 0.05, ***P < 0.001. There was also an association (D) with female sex (F₁ = 4.01, P ≤ 0.05) but no other significant associations owing to generation (F0, F1, and F2) or interactions among any of the independent (lineage, generation, and sex) variables.

Fig. 2.. ASC1, ASC2, and ASC3 transcripts, as revealed by in situ hybridization RNAscope across central and peripheral nervous system structures.

The figure is divided by structure (medulla, PAG, cerebellum, DRG, and cortex) and shown by Asic subtype (Asic1, Asic2, and Asic 3) with nuclear (DAPI) and neuronal (Nissl NeuroTrace) staining.

Fig. 3.. RCF is associated with enhanced ASIC expression in neurons of the MO and PAG matter trigenerationally (data are represented as means + SEM).

(A) Multivariate (n = 38) association between ASIC transcripts and the RCF lineage by multivariate analysis of variance (MANOVA) (Hotelling’s trace, F_3,30 = 4.5, P ≤ 0.01) in the neuronal population of the MO. Tests of between-subjects’ effects showed significant univariate effects for all three ASIC populations (ASIC1: F₅ = 4.00, P ≤ 0.007; ASIC2: F₅ = 2.50, P ≤ 0.05; ASIC3: F₅ = 3.20, P ≤ 0.02) and the significant univariate effect of RCF lineage on all three ASIC populations (ASIC1: F₁ = 11.35, P ≤ 0.002; ASIC2: F₁ = 7.60, P ≤ 0.01; ASIC3: F₁ = 7.25, P ≤ 0.01). There was no multivariate effect of generation [Hotelling’s trace, F_6,58 = 1.2, P = not significant (NS)] or lineage-by-generation interaction; there was no univariate effect of generation on any of the three ASIC subtypes or any significant lineage-by-generation univariate interaction on any of the three ASIC populations. No significant multivariate effects of lineage, generation, or lineage-by-generation interaction were found on ASIC1, ASIC2, or ASIC3 transcripts of nonneuronal MO cells by MANOVA. See also the Supplementary Materials for follow-up analyses of MANOVA results. (B) Multivariate association (n = 47) between ASIC transcripts and the RCF lineage by MANOVA (Hotelling’s trace, F_3,39 = 5.80, P ≤ 0.002) in the neuronal population of the PAG. Tests of between-subjects’ effects showed only the significant univariate effect for ASIC1 (ASIC1: F₅ = 5.60, P ≤ 0.001) and the significant univariate effect of RCF lineage (F₁ = 17.52, P ≤ 0.001) on ASIC1. *P < 0.05, **P < 0.01. There were no multivariate effects of generation (Hotelling’s trace, F_6,76 = 1.60, P = NS) or lineage-by-generation interactions. No significant multivariate effect of lineage, generation, or lineage-by-generation interaction was found on ASIC1, ASIC2, or ASIC3 transcript of nonneuronal PAG cells by MANOVA.

Fig. 4.. Single-dose pretreatment with nebulized amiloride normalizes homeostatic responses in RCF animals trigenerationally (data are represented as means + SEM).

(A) Association (n = 46) between Vt during 6% CO₂ breathing and amiloride/saline pretreatment (F_1,40 = 15.3, P ≤ 0.001) and interaction between RCF lineage and amiloride pretreatment (Hotelling’s trace, F_1,40 = 23.93, P ≤ 0.001) by ANOVA-R. There were no other significant associations owing to generation (F0, F1, and F2) or any possible combinations of the predictors. (B) Association (n = 130) between latency of paw withdrawal at mechanosensitive stimulation (SUDO–von Frey’s test) and the RCF lineage (F₁ = 31.2, P ≤ 0.001), pretreatment with amiloride (F₁ = 15.0, P ≤ 0.001), and the interaction between the former two factors (F₁ = 9.70, P ≤ 0.002) by univariate GLM (F₂₃ = 5.30, P ≤ 0.001). No other significant effects (linear or interactive) emerged from these analyses. (C) Association (n = 152) between latency of paw withdrawal at thermal stimulation (Hargreaves’ test) and the RCF lineage (F₁ = 26.80, P ≤ 0.0001), pretreatment with amiloride (F₁ = 263.14, P ≤ 0.0001), and the interaction between the former two factors (F₁ = 37.00, P ≤ 0.0001) by univariate GLM (F₂₁ = 27.60, P ≤ 0.0001). **P < 0.01, ***P < 0.001. No other significant effects (linear or interactive) emerged from these analyses. See also the Supplementary Materials for follow-up analyses of MANOVA results.