The hyperexcitability of laterodorsal tegmentum cholinergic neurons accompanies adverse behavioral and cognitive outcomes of prenatal stress

Abstract

Exposure to prenatal stress (PS) leads to the offspring's vulnerability towards the development of cognitive and behavioral disorders. Laterodorsal tegmentum (LDT) is a part of the brainstem cholinergic system that is believed to play a pivotal role in the stress-associated progression of anxiety, memory impairment, and addictive behaviors. In this study, we aimed to investigate the electrophysiological alterations of LDT cholinergic neurons and its accompanied behavioral and cognitive outcomes in the offspring of mice exposed to physical or psychological PS. Swiss Webster mice were exposed to physical or psychological stress on the tenth day of gestation. Ex vivo investigations in LDT brain slices of adolescent male offspring were performed to evaluate the effects of two stressor types on the activity of cholinergic neurons. Open field test, elevated plus maze, passive avoidance test, and conditioned place preference were conducted to assess behavioral and cognitive alterations in the offspring. The offspring of both physical and psychological PS-exposed mice exhibited increased locomotor activity, anxiety-like behavior, memory impairment, and preference to morphine. In both early- and late-firing cholinergic neurons of the LDT, stressed groups demonstrated higher firing frequency, lower adaptation ratio, decreased action potential threshold, and therefore increased excitability compared to the control group. The findings of the present study suggest that the hyperexcitability of the cholinergic neurons of LDT might be involved in the development of PS-associated anxiety-like behaviors, drug seeking, and memory impairment.

Figure 1

Timeline of prenatal stress induction, behavioral assays and electrophysiological procedures.

Figure 2

Comparison of the effect of PS on locomotor activity and anxiety-like behavior of the offspring in the open field test. (A) Frequency of grooming per session. (B) Frequency of rearing per session. (C) Total distance moved. (D) Duration of mobility. (E) Mean velocity. (F) Time spent in the center zone. Data are displayed as Mean ± SEM. Phy s: physical stress, Psy s: psychological stress, * (p < 0.05), ** (p < 0.01), *** (p < 0.001) compared to the control group.

Figure 3

Assessment of the effect of PS on anxiety-like behavior of the offspring in EPM. (A, B) Frequency of entries into open and closed arms. (C, D) Time spent in open and closed arms. Data are displayed as Mean ± SEM. Phy s: physical stress, Psy s: psychological stress, * (p < 0.05) compared to the control group.

Figure 4

The effect of PS on passive avoidance learning and memory of the offspring. (A) Number of shocks required for learning. (B) Step through latency. Data are displayed as Mean ± SEM. Phy s: physical stress, Psy s: psychological stress, * (p < 0.05) compared to the control group.

Figure 5

Assessment of the effect of PS on conditioned place preference (CPP) score of the offspring. CPP score is defined as the difference in time spent in the non-preferred chamber on post-conditioning day and the time spent in the non-preferred chamber on pre-conditioning day. Data are displayed as Mean ± SEM. Phy s: physical stress, Psy s: psychological stress, * (p < 0.05) compared to the control group.

Figure 6

Injection of 100 pA positive current for 500 ms after clamping at − 70 Mv in offspring of physical and psychological prenatal stressed mothers. (A) Firing frequency of early-firing neurons. (B) Firing pattern of early- and late- firing neurons. (C) Firing frequency of late-firing neurons. (D) Adaptation ratio of early-firing neurons. (E) Adaptation ratio of late-firing neurons. (F) AP threshold of early-firing neurons. (G) AP pattern of early- and late- firing neurons. (H) AP threshold of late-firing neurons. Data are represented as Mean ± SEM. * (p < 0.05), ** (p < 0.01) shows a significant difference compared to the control group. Phy S: Physical stress, Psy S: Psychological stress.

Figure 7

Early-firing neurons' response pattern to positive current injection pulses with intensities of 60 to 140 pA. Data are represented as Mean ± SEM. * (p < 0.05) showed a significant difference compared to the control group. Phy S: Physical stress, Psy S: Psychological stress.

Figure 8

Response of early-firing neurons (A–C) and late-firing neurons (D, F) to Ramp flow injection pulses (0 pA to 2nA lasting 1000 ms) and their corresponding early (G) and late (H) rheobase. Data are represented as Mean ± SEM. *** (p < 0.001) * (p < 0.05) shows a significant difference compared to the control group. Phy S: Physical stress, Psy S: Psychological stress.