Early-Life Neglect Alters Emotional and Cognitive Behavior in a Sex-Dependent Manner and Reduces Glutamatergic Neuronal Excitability in the Prefrontal Cortex

Abstract

Early-life neglect in critical developmental periods has been associated with emotional and cognitive consequences. Maternal separation (MS) has been commonly used as a rodent model to identify the developmental effects of child neglect. However, reports have shown considerable variability in behavioral results from MS studies in both mice and rats. Difficulties in developing reliable child neglect models have impeded advances in identifying the effects of early-life stress. Accumulating evidence shows that neuronal intrinsic excitability plays an important role in information processing and storage in the brain. The prefrontal cortex (PFC) integrates information from many cortical and subcortical structures. No studies to date have examined the impact of early-life stress on glutamatergic neuronal excitability in the PFC. This study aimed to develop a reliable child neglect rat model and observe glutamatergic neuronal excitability in the PFC. An MS with early weaning (MSEW) rat model was developed. Rats were separated from the dam for 4 h per day on postnatal days (PNDs) 2-5 and for 8 h per day on PNDs 6-16 and then weaned on PND 17. A battery of behavioral tests was used to assess anxiety-like behavior, coping behavior, working memory, spatial reference memory, and fear memory. The action potentials (APs) of glutamatergic neuronal membranes were recorded. MSEW resulted in anxiety-like behavior, a passive coping strategy and increased fear memory in male rats and decreased locomotor activity in both sexes. MSEW slightly impaired working memory during non-stressful situations in female rats but did not change spatial reference memory or associative learning under stressful circumstances in either sex. MSEW reduced the number of glutamatergic neuron APs in male rats. Our findings showed that MS with early weaning induced anxiety-like behavior in male rats. The reduced glutamatergic neuronal excitability may be associated with the emotional alteration induced by MSEW in male rats. In addition, MSEW induced adaptive modification, which depended on a non-stressful context.

Keywords: anxiety like behavior; child neglect; learning and memory; maternal separation; neuronal excitability; rats.

Figure 1

Maternal separation with early weaning (MSEW) induced anxiety-like behavior in the open field test in male rats and decreased locomotor activity in both males and females. MSEW significantly reduced the distance moved and velocity in the open field in both female and male rats (A,B). MSEW increased the index of thigmotaxis in male rats and reduced the center time only in males (C,D). Female and male MSEW rats had increased numbers of feces. (E) All results are presented as the mean ± standard error of the mean (SEM) of n = 8–10 rats per group. **P < 0.01, control vs. MSEW of the same sex; ^##P < 0.01, male vs. female in controls.

Figure 2

Maternal separation with early weaning (MSEW) induced anxiety-like behavior in the elevated plus maze test in male rats and decreased locomotor activity in both sexes. Male MSEW rats exhibited reduced percentage of time spent on the open arms (%OT), head-dipping frequency, distance moved, and total entries (A–D). MSEW reduced the time in the center area and increased the time in the closed arms in male rats (E,F). All results are presented as the mean ± standard error of the mean (SEM) of n = 8–10 rats per group. **P < 0.01, control vs. MSEW of the same sex; *P < 0.05, **P < 0.01, control vs. MSEW of the same sex.

Figure 3

Maternal separation with early weaning (MSEW) slightly impaired non-spatial working memory in the novel object recognition test in female rats. The total exploration time did not differ between the control and MSEW groups of the same sex, and female control rats showed a longer exploration time than male control rats (A). During the test session (2 h later), female MSEW rats showed significantly reduced discrimination index (d1) but no change in the d1 compared to the control females (B,C). Male MSEW rats showed very low exploration levels of both the novel object and the familiar object (D). All results are presented as the mean ± standard error of the mean (SEM) of n = 8–10 rats per group. *P < 0.05, control vs. MSEW of the same sex; ^##P < 0.01, male vs. female in controls.

Figure 4

Maternal separation with early weaning (MSEW) slightly impaired spatial working memory in the Y maze in female rats. Female control rats entered the novel arm more than the other arm (A). Female MSEW rats also showed a preference for the novel arm, but the difference was not significant (A). Both control and MSEW male rats entered the novel arm more than the other arm (B). All results are presented as the mean ± standard error of the mean (SEM) of n = 8–10 rats per group. *P < 0.05, **P < 0.01, control vs. MSEW of the same sex.

Figure 5

Maternal separation with early weaning (MSEW) did not impair spatial reference memory in either sex. Compared with control females, MSEW female rats had a tendency to spend more time finding the platform (latency) on the 1st and 2nd days and spent similar time on the 3rd day (A). MSEW and control male rats spent similar amounts of time finding the platform (B). For the probe test, MSEW rats showed no significant difference compared to their respective controls in latency to the target hole, time to the target hole, velocity, or distance moved for either sex (C–F). All results are presented as the mean ± standard error of the mean (SEM) of n = 8–10 rats per group.

Figure 6

Maternal separation with early weaning (MSEW) induced a passive coping strategy in male rats. MSEW increased the immobility time in males. (A) MSEW did not change the climbing time in either sex. (B) All results are presented as the mean ± the standard error of the mean (SEM) of n = 8–10 rats per group. *P < 0.05, control vs. MSEW of the same sex.

Figure 7

Maternal separation with early weaning (MSEW) did not impair associative learning in either sex or increase fear memory in male rats. MSEW male rats showed more freezing behavior than control male rats (A). Female and male MSEW rats did not show impaired associative learning (B). MSEW male rats exhibited increased freezing time compared to the control male rats (C). All results are presented as the mean ± the standard error of the mean (SEM) of n = 8–10 rats per group. *P < 0.05, control vs. MSEW of the same sex.

Figure 8

Maternal separation with early weaning (MSEW) reduced glutamatergic neuronal excitability in the prefrontal cortex in male rats. MSEW did not change the threshold or amplitude of single action potentials in the prefrontal cortex in either male or female rats (A,B). MSEW decreased the half-spike time in female rats (C). Afterhyperpolarization (AHP) in the four groups (D). Representative traces of glutamatergic neurons (150 pA, 600 ms) (E). MSEW male rats had decreased numbers of glutamatergic neuron action potentials (APs) with current injections of 400 and 500 pA (F). Representative traces obtained in the prefrontal cortex neurons from control and MSEW male rats (G,H). Input resistance in the four groups (I). MSEW female rats showed a trend for decreased numbers of glutamatergic neuron APs (J). Representive traces obtained in the prefrontal cortex neurons from control and MSEW female rats (K,L). All results are presented as the mean ± standard error of the mean (SEM) of n = 3–5 rats per group. *P < 0.05, **P < 0.01, control vs. MSEW of the same sex.