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Prenatal Stress on Progeny


The following account of methods is described as done by Soares-Cunha et al. (2018).


Twelve female Wistar han rats were impregnated using six male Wistar han rats. The female rats were virgins, 8-10 weeks old, and weighed approximately 250 g. The male rats were experienced, 6 months old, and weighed approximately 500 g. Impregnation occurred through double-housing two females with one male overnight. These rats were then maintained under standard laboratory conditions, consisting of an ambient temperature of 21 ± 1 ͦ C, a relative humidity of 50%-60%, and an artificial 12-h light/dark cycle (with the lights being on from 08:00 to 20:00). The rats were given water and food, which was a standard mating/pregnancy diet (4rF25, Mucedola SRL), ad libitum. The female rats were determined pregnant once sperm was discovered on their vaginal smear. Once the female rat was classified as pregnant, they were individually housed.

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The subjects used for the behavioural tasks were the progeny of the rats described above. The subjects were therefore Wistar han rats, of both sexes. The progeny was separated from their litter on post-natal day 21, then double-housed with a same-sex, randomly chosen roommate. They were maintained under standard laboratory conditions, consisting of an artificial 12-h light/dark cycle (lights on from 08:00 to 20:00), and a room temperature of 22  ͦ C. The rats were given water and fed 4RF21 and Mucedola SRL ad libitum. To control for potential litter effects, all the progeny from a minimum of four different litters were used in all tests, behavioural and neurochemical. Moreover, all progeny was used in all behavioural tests, and were conducted when the rats were ages 61 days to 115 days. For count of subjects, refer to Table 1.

All animal health handling followed the guidelines of FELASA, and all procedural

handlings followed European regulations. Everyone directly involved with animal handling were certified with the Portuguese regulatory entity. All protocols were approved by the Ethics Committee of ICVS.


Refer to Figures 1 and 2 to follow the procedural timeline.

The behavioural tests that were conducted between 09:00 and 13:00 include the Elevated Plus Maze (EPM), the Open Field (OF), the Light/Dark Box test (LDB), the Forced Swimming Test (FST), the Variable Delay-to-Signal (VDS), and the Progressive Ratio (PR). The two training sessions for VDS and PR were conducted between 09:00 and 19:00, with a minimum of 4 hours between training sessions. The Sucrose Preference Test (SPT) was conducted between 21:00 and 22:00.

Prenatal Stress Protocol

Out of the twelve pregnant female rats, six were randomly selected to an unpredictable, prenatal stress protocol (PS group, = 6). This occurred from pregnancy day 3 to day 20, at a rate of three times a week. The stress protocol either consisted of 4 hours of restraint in a cylindric box 12 cm in diameter, Strobeyellow stroboscopic lights (Disco Pro Light), or exposure to 80 dB noise. Both the stress protocol and time of the week were randomly selected. The control rats remained unperturbed in their cages (CTR group, n = 6).

Behavioural Tests

Anxiety Evaluation.

The anxiety levels of the progeny were evaluated in the following ways:

Open Field (OF).

Rats were placed in the center of a 43.2 cm x 43.2 cm square arena, which had

transparent acrylic walls and a white floor. Their movement within the area was monitored for 10 minutes using two 16-beam infrared arrays. Their total distance travelled was recorded, and used to indicate their locomotor activity.

Elevated Plus Maze (EPM).

Rats were placed in the center of a plus-shaped platform, composed of two open arms (50.8 cm x 10.2 cm) and two closed arms (50.8 cm x 10.2 cm x 40.6 cm). The platform consisted of black polypropylene material, was 72.4 cm above floor-level, and was under bright white lights. The animals had 5 minutes to explore the platform; their time spent in each arm and number of entries into each arm were recorded.

Light/Dark Box (LDB).

One side of the OF arena (43.2 cm x 43.2 cm) was darkened through attaching a dark compartment to one side of the area, with an opening facing the center. Rats were placed in the center of the light portion of the arena. Their movement within the area was monitored for 10 minutes, and their distance travelled and time spent in each compartment was recorded.

Depressive-Like Behaviour Evaluation.

The depressive levels of the progeny were evaluated in the following ways:

Forced Swimming Test (FST).

Rats were forced to swim in cylinders filled with warm water. They were given one pre-test session for 5 minutes, then after 24 hours, retested again for 5 minutes. They were then placed on a heating pad, and returned to their cages. A video camera was used to record their movement, and a blind experimenter determined the amount of time the rat was immobile (or staying in one location), the rat’s number of attempts to climb the walls, and their latency to immobility.

Sucrose Preference Test (SPT).

The rats were tested twice, with a 2-day interval between each trial. 12 hours before each trial, the rats were deprived of both food and water. Then, the individually-housed rat was given two pre-weight bottles for 1 hour: one with water and one with a 2% (m/v) sucrose solution. The rat’s preference for sucrose (SP) was calculated through the following formula: SP = (sucrose intake/(sucrose intake + water intake)) x 100. Anhedonia, the rat’s inability to feel pleasure, was operationally defined as the rat’s reduced preference for sucrose (SP) compared to the control.

Impulsivity Evaluation.

The impulsivity levels of the progeny were evaluated in the following way:

Variable Delay-to-Signal (VDS).

The rats were placed in an operant chamber (25 cm x 25 cm) with five square holes (2.5 cm x 2.5 cm) and a 3W light bulb. There were four chambers being used at the same time; to cancel out noise, each chamber was placed in sound attenuating chambers with white noise. The VDS test spanned 30 minutes, and involved multiple trials in which the rat poked his nose into one of the openings in the chamber. After the nose poke, there was a variable delay, followed by a sugared reward and a simultaneous light illumination (Leite-Almeida et al., 2013). To record the rat’s movements, multiple infrared photo beams were placed on a wall. On the wall opposite to it, there was an aperture connected to a food dispenser, with lights and photobeams. This wall additionally held the house light illuminating the arena.

The entire protocol included trials of habituation, training, and testing, all in which the

rats were food-deprived. The procedure is as follows; first, the rats spent 2 days habituating. Then, for five consecutive days, the rats received two training sessions a day with a 5-hour interval in between sessions. These training sessions consisted of turning on the house light and distributing one sugar pellet with an intertrial interval (ITI) of 3 seconds. The trials had a delay period of 3 seconds where the house light remained on, followed by a response period of 60 seconds in which the hole light is on. If the rat stuck its nose in the hole during the delay period, they were punished by a timeout period with no lights. If the nose pokes during the response period, the rats received a pellet, followed by a 3-second ITI, then a new trial. These training sessions continued until the rat either completed 100 trials, or 30 minutes have elapsed. Any rat that could not complete 100 trials within 30 minutes by the end of the training period was excluded from further testing. Finally, the rats were tested. Testing took place in one day, and consisted of 120 trials. The test used the same protocol as the training session, except for a 3-second delay in the first and last 25 trials, and a 6- or 12-second delay in the middle 70 trials.

Motivation Test.

The motivation levels of the progeny were evaluated in the following way:

Progressive Ratio (PR) Schedule of Reinforcement.

Rats were first put on a food restriction diet, being given around 7 g of standard lab chow a day. Then, to familiarize the rats to their motivation (food pellets), 45 mg of food pellets were placed in their cages before training.

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The actual behavioural tests were conducted in operant chambers, and followed previous methods (Wanat et al., 2010; Soares-Cunha et al., 2016). For training, the rats spent 6 days in continuous reinforcement (CRF) training, in which pressing on a lever immediately rewarded the rat with one food pellet. This was followed by one session of fixed-ratio (FR) 1 training, in which pressing the lever once rewarded the rat with one food pellet, then four sessions of FR4 (four lever presses for one food pellet), and one session of FR8 (eight lever presses for one food pellet). Actual testing lasted a maximum of 15 minutes. Their response requirement was identical to the FR4 session, however their requirement on each trial (T) was the integer (rounded down) of 1.4(T-1) lever presses (starting at one lever press). This directly tests motivation, as it determines the amount of work a rat is willing to do for a reward.

Physiological and Neurochemical Tests

Corticosterone Measurement.

The corticosterone levels of the mothers were determined through collecting 250 μl of blood from their tails four times: at 8:00 and 20:00 before prenatal stress, and at 8:00 and 20:00 after the final prenatal stress procedure. The corticosterone levels of the progeny were determined from collecting 250 μl of blood from their tails 30 minutes before an acute stress protocol, and 120 minutes after. At least four different litters of progeny were tested.

The concentration of corticosterone was determined through an ELISA corticosterone kit; this involved extracting 10 μl of plasma from the sample and testing it through the corticosterone assay. Quantification was determined from the average net optical density.

Corticosterone Levels in Response to Acute Stress.

To test the corticosterone response from the HPA axis in acute stress, blood samples were taken from the rat’s tails before and after an acute stressor. The blood sample was first drawn at 14:00. This was followed by an acute stressor (30-minute restraint protocol). Two hours after this stressor, blood was collected again. These tests were conducted on the pregnant mothers on gestation day 20, and on the progeny on post-natal day 60.


The progeny of both control and PS rats (n = 6) were anesthetized with a lethal dose of pentobarbital. The experimenters then cut off the rat’s heads, and froze it in liquid nitrogen. Specific brain regions were dissected on ice through a stereomicroscope, then stored in −80°C.

Neurochemical Evaluation.

To evaluate levels of catecholamines, the experimenters used a Gilson instrument with an analytical column. This was used to conduct high-performance liquid chromatography with electrochemical detection. The resulting samples were then stored overnight in 0.2 N perchloric acid at −20°C, spent 5 minutes on ice, then centrifuged at 5000 g. This solution was then filtered through a Spin-X HPLC column, and 150 mL of aliquots were injected using a mobile phase of 0.7 M aqueous potassium phosphate (pH 3.0) in 10% methanol, 1-heptanesulfonic acid (222 mg l−1) and Na-EDTA (40 mg l−1).

Histological Procedures.

The progeny of both control and PS rats from at least 4 different litters (n = 4) were anesthetized with pentobarbital and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). The rat’s brains were then removed from their body, and placed in 4% PFA for four weeks. The experiments then cut the brain midsagittally, and prepared it for stereology; this consisted of temporarily putting the brain in glycolmethacrylate (Tecnovit 7100), then collecting the other microtome-cut sections (30 μm wide) on a gelatinized slide, stained it with Giemsa, and mounted it with Entellan New.

Stereological Procedures.

Materials used to estimate volume include the StereoInvestigator software, a motorized

microscope, and a Sony DXC-390 camera; the Cavalieri’s principle (Gundersen et al., 1999) was used to evaluate this volume.

For the following brain regions, every 4th section was used: BNST, central amygdala (CeA), and basolateral amygdala. For the following brain regions, every 8th section was used: NAc core and NAc shell. For the following brain regions, every 12th section was used: CA1, CA3, and dentate gyrus.

The optical fractionator method (West et al., 1991) was used to estimate the total number of cells; this consisted of using a grid of virtual 3D-boxes (30 μm × 30 μm × 15 μm) superimposed on every laminal section of interest, then counting the cells within each grid box. Glial cells were not counted. The coefficients of error for cell numbers was calculated based on Gundersen et al. (1999)’s formula, and for volume estimations, Gundersen and Jensen (1987)’s formula.

Statistical Analysis

To eliminate possible outliers, the Shapiro-Wilk test and Tukey’s fences were conducted on all data. The Student’s t-test analyzed corticosterone levels, stereology data, catecholamine levels, and every behavioural test except the VDS, and the training sessions for the PR test. The CRF and FR training sessions for the PR test performed an analysis of variance (ANOVA) and Bonferroni post-hoc test. A Mann-Whitney test was used for the abnormal, non-parametric catecholamine levels, which occurred in the BNST distribution.

Results are described as a mean ± SEM, and were considered significant if p ≤ 0.05. Statistical analysis was conducted using Prism GraphPad (v7).


  • Gundersen, H. J., and Jensen, E. B. (1987). The efficiency of systematic sampling in stereology and its prediction. Journal of Microscopy, 147, 229–263.
  • Gundersen, H. J., Jensen, E. B., Kiêu, K., and Nielsen, J. (1999). The efficiency of systematic sampling in stereology–reconsidered. Journal of Microscopy, 193, 199–211.
  • Leite-Almeida, H., Melo, A., Pêgo, J. M., Bernardo, S., Milhazes, N., Borges, F., et al. (2013). Variable delay-to-signal: a fast paradigm for assessment of aspects of impulsivity in rats. Frontiers in Behavioral Neuroscience, 7(154).
  • Soares-Cunha, C., Coimbra, B., David-Pereira, A., Borges, S., Pinto, L., Costa, P., et al. (2016). Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation. Nature Communications, 7.
  • Soares-Cunha, C., Coimbra, B., Borges, S., Domingues, A. V., Silva, D., Sousa, N., & Rodrigues, A. J. (2018). Mild prenatal stress causes emotional and brain structural modifications in rats of both sexes. Frontiers in Behavioral Neuroscience, 12, 1-15.
  • Wanat, M. J., Kuhnen, C. M., and Phillips, P. E. M. (2010). Delays conferred by escalating costs modulate dopamine release to rewards but not their predictors. Journal of Neuroscience, 30, 12020–12027.
  • West, M. J., Slomianka, L., and Gundersen, H. J. (1991). Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. The Anatomical Record, 231, 482–497.


Table 1

Copied from Soares-Cunha et al. (2018). A table showing the number of offspring in a litter, and

their sex. Showing count of both control and PS mothers.


Figure 1. Copied from Soares-Cunha et al. (2018). The timeline of the experiment.

Figure 2. Copied from Soares-Cunha et al. (2018). Timeline of the behavioural tests; all

progeny were tested, and all in this order.


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