Female C57Bl/6 mice (postnatal day > 60) from Fundação Oswaldo Cruz (Fiocruz) breeding colony were used. Animals were group housed (maximum 5 per cage), in a 12/12 light-dark cycle room, with free access to standard food and tap water. All methods and procedures were carried out under the guidelines established by Colégio Brasileiro de Experimentação Animal (COBEA) and the Guidelines on the Care and Use of Animals for Experimental Purposes and Infectious Agents (NACLAR). All experimental protocols were approved by Fundação Oswaldo Cruz - Fiocruz Committee of Ethics for the Use of Animals (license L042/18 A1).
Parasites and Experimentally Acquired Toxoplasmosis
T. gondii of ME-49 strain (Type II) were used and maintained in C57BL/6 female mice, weighing about 12–18 g each. For infection, parasites were inoculated intraperitoneally with about 30 cysts/animals diluted in 200 μL of phosphate-buffered saline (PBS), while control animals were injected with PBS only. Animals were euthanized with ketamine the following day after behavioral tests. Their brains were collected, prefrontal cortices and hippocampi were dissected and macerated to determine neurotransmitters, as described in detail below.
Mice were behaviorally tested 8 weeks after infection using spontaneous alternations, tail suspension, prepulse inhibition of the startle reflex (PPI) and social approach tests. Behavioral tasks were performed during the light cycle, between 8:00 am and 5:00 pm in a dimly lit room with minimal background noise. All test apparatuses were cleaned with 10% ethanol between each session and with 70% ethanol at the end of the last session of experiments.
Mice were allowed to freely explore a Y maze (40 x 8 x 20 cm each arm) for eight minutes. The total number and sequence of arms entrances were recorded. A spontaneous alternation is characterized when the animal enters three different arms in sequence. The percentage of spontaneous alternations was calculated as a measure of working memory integrity. The position bias index of each animal was calculated to investigate motor impairments' interference in the task (33). This index indicates whether animals show a trend for always turning left or turning right in the maze, which might compromise results interpretation.
Tail suspension test
The tail suspension test was conducted to investigate the development of depressive-like behavior. In this test, mice tend to develop an immobile posture when placed in an inescapably stressful situation after initial escape-oriented movements. Mice were individually suspended (using an adhesive tape placed 1 cm from the tip of the tail) about 65 cm above the floor for 6 minutes. Immobility was recorded only when mice hung passively and completely motionless. Depression-like behavior shown by the time spent immobile was measured in the last 4 min of the 6-min-long test as previously described (34). Sessions were videotaped and the immobility time (s) was blindly scored.
Prepulse inhibition of the startle reflex (PPI)
PPI test was performed to investigate the sensorimotor gating and was conducted as previously described (35). Briefly, a startle box system (Panlab®) containing a sound generator and an accelerometer was used to record the amplitude of mice’s startle response (ASR). A white background noise (65 dB) was generated throughout the experiment. Mice were kept in the chamber for five minutes of habituation, and then five blocks containing a startle-inducing pulse (white noise, 120 dB, 50 ms) were presented. After that, mice were randomly exposed to 5 different blocks of stimuli (10 repetitions each): background noise, pulse (white noise, 120 dB, 50 ms), and pulse preceded by prepulse in three different intensities (72, 80 and 90 dB, white noise, 20 ms, 100 ms interstimulus interval). The interval between blocks was 20 ± 10 s. Mice ASR in each block was digitized and recorded (Startle v. 1.2.04, Panlab®). The percentage of startle reflex inhibition at different prepulse intensities was calculated using the following formula: %PPI = 100 - [100 × (ASR mean to prepulse+pulse trials/ASR mean to pulse alone trials)].
Social approach task
Sociability assessment was conducted as previously described (36), with minor modifications. A rectangular transparent plexiglass box (60 x 45 x 30 cm) divided into three equal compartments (20 x 45 x 30 cm) was used. Openings between compartments allow the animal to move from one compartment to another freely. Cylindrical aluminum cages (9.5 cm high x 8 cm diameter) were used to contain stimuli mice. Test mice were habituated in the three-chamber apparatus in the presence of empty cages, one in each lateral chamber, for 15 minutes one day before the test. On the same day, social stimuli (conspecific adult female mice) were habituated inside cylindrical cages for two sessions of 30 minutes. In the social approach test, a social stimulus was placed inside a cage in one of the lateral compartments while an identical empty cage was placed in the opposite compartment. Test mice were initially placed in the central compartment without access to the lateral compartments for 5 minutes. After that, blocking walls were removed and test mice could move between compartments for 10 minutes. Sessions were videotaped and interaction time with the both empty and social cages (s), permanence time and the number of entries in each compartment were analyzed.
Measurement of aminoacids levels
After the infection period, animals were euthanized, brains were removed and the prefrontal cortex and hippocampus of each animal were rapidly dissected. Tissue fragments were homogenized at 4 °C in RIPA buffer (Sigma, St. Louis, USA). We analyzed glutamate, l-serine, glutamine, and D-serine levels by high-performance liquid chromatography (HPLC) as previously described (37, 38).
SigmaStat version 3.01 (Jandel Scientific Corporation®) or GraphPad Prism® version 6.01 softwares were used. Unpaired Student’s t-test was performed to analyze data between experimental groups (control and infected). In the social approach test, the percentage of interaction time with the social cages was analyzed using a one-sample Student's t-test using a 50% value as standard, while the data that contains the percentage of time spent in each chamber of the apparatus was analyzed by two-way ANOVA followed by Bonferroni-Sídák test for multiple comparisons. % PPI data were analyzed by two-way repeated-measures ANOVA with experimental groups as the first factor and prepulse intensity as the second one. This analysis was followed by Tukey’s test for multiple comparisons. Significance level p <0.05 was considered significant. All experimenters were blind to control and experimental groups.