Glucocorticoid programing of the mesopontine cholinergic system

Abstract

Stress perception, response, adaptation, and coping strategies are individually distinct, and the sequel of stress and/or glucocorticoids (GCs) is also distinct between subjects. In the last years, it has become clear that early life stress is a powerful modulator of neuroendocrine stress-responsive circuits, programing intrinsic susceptibility to stress, and potentiating the appearance of stress-related disorders such as depression, anxiety, and addiction. Herein we were interested in understanding how early life experiences reset the normal processing of negative stimuli, leading to emotional dysfunction. Animals prenatally exposed to GCs (in utero glucocorticoid exposure, iuGC) present hyperanxiety, increased fear behavior, and hyper-reactivity to negative stimuli. In parallel, we found a remarkable increase in the number of aversive 22 kHz ultrasonic vocalizations in response to an aversive cue. Considering the suggested role of the mesopontine tegmentum cholinergic pathway, arising from the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT), in the initiation of 22 kHz vocalizations and hypothetically in the control of emotional arousal and tone, we decided to evaluate the condition of this circuit in iuGC animals. Notably, in a basal situation, iuGC animals present increased choline acetyltransferase (ChAT) expression in the LDT and PPT, but not in other cholinergic nuclei, namely in the nucleus basalis of Meynert. In addition, and in accordance with the amplified response to an adverse stimulus of iuGC animals, we found marked changes in the cholinergic activation pattern of LDT and PPT regions. Altogether, our results suggest a specific cholinergic pathway programing by prenatal GC, and hint that this may be of relevance in setting individual stress vulnerability threshold.

Keywords: acetylcholine; anxiety; fear; glucocorticoids; laterodorsal tegmental nucleus; pedunculopontine tegmental nucleus; stress; ultrasonic vocalizations.

Figure 1

In utero glucocorticoid exposure induces an anxious-like behavior. iuGC group presented a decrease in ambulatory counts (A), total distance traveled (B), and percentage of time in the center of the arena (C). In the EPM, iuGC animals exhibited a decrease in the percentage of time spent (D) and number of entries (E) in the open arms of the maze, when compared with control group. (F) In the L/D test, iuGC presented an increase in the ratio dark/light, with no differences on the distance traveled in both compartments (G). In the confined cage, iuGC group presented an increase in the number of 22 kHz USVs (H) and freezing behavior (I). Upon habituation, iuGC animals no longer presented this anxious behavior (day 2). CONT, control animals; iuGC, in utero GC exposed animals; USVs, ultrasonic vocalizations. *p < 0.05, **p < 0.001, and ***p < 0.0001; (A–C) n = 16; (D,E) n = 11–13; (F,G) n = 10, (H,I) n = 8–16.

Figure 2

Prenatal exposure to GC leads to amplified response to adverse stimulus. (A) Number of 22 kHz USVs on the conditioning day (cue + shock) of the fear-conditioning paradigm. iuGC group emitted more negative calls than control animals in the light/shock period. (B) Similarly, iuGC group displayed increased percentage of freezing behavior in comparison with control animals. On the test day, animals were exposed six times to cue but no shock was given. iuGC presented an increase in the number of USVs (C) and in freezing time (D). (E) iuGC animals are hyper-reactive throughout time, since they emit more context-induced 22 kHz vocalizations than control group, especially in the first 60 s. Upon first cue exposure (ON period), both groups increased the number of USVs with no major differences between them. However, upon second cue exposure, iuGC animals emitted more negative USVs than controls and remained over-reactive during time. CONT, control animals; iuGC, in utero GC exposed animals; USVs, ultrasonic vocalizations. *p < 0.05, **p < 0.001, ***p < 0.0001, δ: trend, p = 0.130; (A–E) n = 8–16.

Figure 3

Mesopontine cholinergic de-regulation in iuGC animals. (A) LDT ChAT immunohistochemistry revealed that iuGC animals presented increased ChAT expression (B). (C,D) Similarly, iuGC animals present increased ChAT staining in the PPT region. Conversely, no differences were found in ChAT expression in the nucleus basalis of Meynert (E,F) or in the nucleus accumbens core or shell (G,H). Representative images of coronal brain sections; numbers represent distance in mm posterior to bregma. CONT, control animals; iuGC, in utero GC exposed animals; LDT, laterodorsal tegmental nucleus; NAc, nucleus accumbens; PPT, pedunculopontine tegmental nucleus. *p < 0.05, **p < 0.001, ***p < 0.0001; (A) n = 4–7. Scale bars: 50 μm.

Figure 4

Stereological data of LDT and PPT regions. No major differences were observed in the volumes and cell numbers of LDT (A–C) and PPT regions (D–F) of iuGC animals when compared to controls. Representative images of coronal brain sections; numbers represent distance in millimeters posterior to bregma. CONT, control animals; iuGC, in utero GC exposed animals; LDT, laterodorsal tegmental nucleus; PPT, pedunculopontine tegmental nucleus. (B,C) n = 5–6; (E,F) n = 4.

Figure 5

Differential cholinergic activation of the LDT and PPT regions in iuGC animals after an adverse stimulus. (A) Representative images of LDT ChAT (red labeling) and c-fos (green labeling) immunostaining in animals exposed to the fear-conditioning test. (B) Increased c-fos activation in iuGC animals. (C) ChAT expression was increased in iuGC animals in comparison to controls. (D) The number of c-fos/ChAT-positive cells was substantially increased in the LDT of iuGC animals. (E) Representative images of PPT ChAT and c-fos immunostaining in animals exposed to the fear-conditioning test. (F) Increased c-fos staining in iuGC animals. (G) ChAT expression was augmented in iuGC animals. (H) The number of c-fos/ChAT-positive cells is significantly increased in the PPT of iuGC group. CONT, control animals; iuGC, in utero GC exposed animals; LDT, laterodorsal tegmental nucleus; PPT, pedunculopontine tegmental nucleus. *p < 0.05, **p < 0.001; (A–H) n = 6. Scale bars: 50 μm.