Maternal Stress Combined with Terbutaline Leads to Comorbid Autistic-Like Behavior and Epilepsy in a Rat Model

Animals and treatment.

Female Sprague-Dawley rats (n = 12, Harlan Laboratories) 220–245 g at embryonic day (E)2 were allowed to acclimate to the animal facility for 2 d before experimental manipulation. The pregnant dams were individually housed in a sealed and vented cage (Sealed Space Plus, Techniplast). Animals were housed under standard laboratory conditions in a temperature 20°C ± 1, and relative humidity 60% controlled environment, with a normal 12 h light/dark cycle (lights on 7:00 A.M.–7:00 P.M.). Food and water were available ad libitum. All procedures were performed in accordance with University of Colorado Institutional Animal Care and Use Committee guidelines for the humane use of laboratory rats in biological research.

Dams were randomly assigned to either a treatment group receiving prenatal stress (n = 5) or to a control nonstressed group (n = 4). To avoid litter size confounds due to a high mortality rate in the animals receiving both stress and terbutaline, pregnant dams were exposed to a daily mild prenatal stress paradigm from E3 through E21. On E3, each pregnant dam was placed in a sound-attenuated chamber for a 5 min exploration in the novel environment. To establish contextual fear conditioning, on E4 each dam was placed in the same environment and after an initial 60 s exposure, two7 mA shocks (60 s intershock interval) were delivered. The dam was left in the chamber for a 5 min post-shock period. Dams were then replaced in the conditioning chamber for a 5 min re-exposure to the aversive environment for a subsequent 5 d of extinction. The paradigm was repeated weekly through out pregnancy to reinstate and maintain mild stress. Maternal weights were also recorded at E3, E9, E15, and E21, as well as the presence of nests in their home cage on P21 as a measure of stress. The control pregnant females were left undisturbed in their home cage.

Pups from all litters were randomized the day after birth and redistributed to the dams within their own treatment group. To ensure standardized nutrition and maternal care litter size was culled to 10 male pups per dam, allowing each dam to foster no more than two pups from their own litter. The litters were then divided into four treatment groups: controls (no maternal stress and only saline injections for the pups; “noSt+Sa”), those receiving terbutaline alone (“noSt+TB”), maternal stress alone (“St+Sa”), and those receiving the combination of maternal stress followed by terbutaline (“St+TB”). To enable comparison across all four groups, all animals were given either terbutaline sulfate (Sigma-Aldrich) or equivalent volumes of saline, administered daily on postnatal days (P)2–P5. For tocolytic therapy in humans, terbutaline doses typically lie in the range of 0.5 mg/kg per day, but can be as high as 1–2 mg/kg per day (Lam et al., 1998). In light of the fact that terbutaline has a much shorter half-life in the rat (Tegnér et al., 1984), terbutaline was administered in doses of (10 mg/kg), replicating other rat studies (Zerrate et al., 2007; Witter et al., 2009; Slotkin and Seidler, 2013) of this compound. Because the rat is an altricial species, P2–P5 injections have been approximated to be equivalent to the second (Rodier, 1988; Rice and Barone, 2000; Clancy et al., 2001; Zerrate et al., 2007) or third (Avishai-Eliner et al., 2002) trimester of human development, both periods of terbutaline administration where detrimental developmental consequences could be expected (Rhodes et al., 2004; Perna et al., 2014).

Repetitive behavior.

P35: grooming was evaluated by placing the animal in a circular arena (130 cm in diameter) 5 min. A blind observer scored the videos and recorded the time spent grooming for all animals.

Social exploration.

Social exploration tests of anxiety (File and Seth, 2003) were conducted as described previously (Christianson et al., 2011). All animals were tested in a randomized order. At P40, treatment animals were placed in plastic tub cage (45 × 22 cm) with 2 cm of fresh bedding located in a designated testing room for a 1 h habituation period. A conspecific juvenile rat was then introduced into the cage for 3 min. The behavioral response (of the pup) to the juvenile was recorded with a video camera placed above the cage. The behavior was then assessed for each individual animal by measuring total duration and frequency of social exploration.

Ultrasonic vocalizations at P6 and P60.

Ultrasonic vocalizations (USVs) were measured at P6 and P60. A wide frequency range (15–180 kHz) microphone/preamplifier was centered 10 cm above a plastic container (10 × 10 cm) located in a sound-attenuated chamber. USVs were digitized at 1 mHz and analyzed for USV duration using custom-written software. At P6, each pup was taken directly from its home cage, placed in the recording chamber, and USVs recorded for 3 min. Each pup was then weighed and placed back its home cage. At P60, each animal was placed in the recording chamber, and after a 2 min habituation period, presented with two bursts of white noise (30 ms, 110 db, 2 s interval). USVs were recorded for 5 min following the last stimulus.

Composite ASD score.

Because ASD-like behavior is comprised of deficits in the combined core behaviors examined here, we computed an ASD score (similar to the autism severity score put forward by El-Kordi et al., 2013) that reflected a combination of the behavioral tests. All scores across rats within a given test were first converted to Z-scores (demeaned and divided by SD) to be of comparable magnitude between tests. Z-scores for USVs at 6 and 60 d as well as Z-scores for social exploration were inverted so that, when combined with grooming, greater ASD-like behavior would be consistently reflected in a higher score. Scores for the four tests were then averaged to form a composite ASD score. For graphical purposes, the composite ASD scores were scaled to a range of 0–1 by subtracting the minimum value and dividing by the subsequent maximum value.

Chronic video/EEG recording.

At ∼2 months of age, after all behavioral data were collected and ASD-like behaviors were assessed, five rats from each group were instrumented for chronic video/EEG recording. Aseptic surgical procedures were used for all chronic preparations. Under isoflurane (Abbott Laboratories) anesthesia (2.5%), rat pups (2 months of age; 275–300 g) were implanted with a Teflon-coated electrode (in an auditory responsive zone of the hippocampus; AP: −3.8 mm, ML: 1.1 mm, DV: 3.8 mm), a parietal cortical screw electrode (AP: −2 mm, ML: 2 mm), a ground screw (AP: −1 mm, ML: 1 mm), and a reference screw over the occipital bone. Buprenorphin (0.1 mg/kg, subcutaneously) was administered immediately after the surgical procedure and again every 12 h for a 72 h period. Following a 2 week recovery period, animals were tethered to an electrode harness (Plastics One, 363) and a slip-ring commutator (Plastics One, SL6C) permitted free movement for 24/7 video/EEG monitoring throughout the duration of the experiment. Spontaneous EEG signals were amplified and digitized at 500 Hz and stored in 30 min segments with time-locked video records for subsequent seizure and interictal spike analysis. Because seizures were detected in rats of the St+TB group in this initial experiment, four additional St+TB pups were prepared and recorded in a second experiment to replicate the epilepsy results.

Convulsive seizure detection and quantification.

Visual detection of convulsive seizures was performed using custom software. All video/EEG recorded 24/7 for a given rat was displayed in 30 min blocks on high-resolution monitors. All ictal-like events were examined with corresponding video to distinguish convulsive activity from potential animal-generated noise, such as eating and grooming. Verified seizures were of focal origin and secondarily generalized, and convulsive intensity was rated according to Racine's behavioral scale (Racine, 1972). Seizure onset times and behavioral intensities were digitally logged. EEG was simultaneously examined for interictal spikes distinct from convulsive seizures.

Interictal spike detection and quantification.

Supervised pattern recognition was used to quantify interictal spikes (IISs), but did not replace visual examination of all video/EEG to detect seizures. In rats where IISs could be visually identified, 20–30 were digitally marked, and then averaged to form an individualized template for each animal. This template was moved through all raw EEG data for that animal and the covariance between template and signal computed in time increments of 1 ms. When the covariance exceeded an operator-determined threshold, the putative IIS was extracted from the raw data and added to a separate spike log for further analysis. Template matching methods such as this are subject to numerous false-positives. For this reason, we trained a support vector machine (SVM; Orrù et al., 2012) to automatically discriminate between IISs and noise (including seizures). Training was done iteratively, and at each iteration, all putative spikes (aligned to the spike waveform peak ±150 ms) were displayed on a high-resolution monitor and a subset of ∼10 correctly and incorrectly identified events were labeled and used to initially train the SVM model. This model then classified all of the spikes, which were redisplayed with their tentative classification and another subset of correctly and incorrectly identified spikes labeled for refinement of the SVM model. Typically within 2–3 iterations, the SVM correctly accepted >90% of the actual spikes and rejected all previously misidentified events.

Immunohistochemistry.

Rats were intracardially perfused with 0.9% saline followed by 4% paraformaldehyde, tissue was collected and stored at −80°C. Tissue was sectioned at 20 μm and mounted onto SuperFrost Plus slides (Fisher Scientific) using a cryostat. Reactive astrogliosis in the hippocampus was assessed with an astrocyte marker (glial fibrillary acidic protein; GFAP), using an avidin-biotin-horseradish peroxidase (ABC) reaction. Sections were treated with 0.3% H₂O₂ for 30 min at room temperature, washed in PBS, and incubated at 4°C overnight in mouse anti-GFAP (1:100; MP Biomedicals). The next day, sections were incubated at room temperature for 2 h with biotinylated goat anti-mouse IgG antibody (1:200; Jackson ImmunoResearch). Sections were then washed and incubated for 2 h at room temperature in ABC (1:400 Vector Laboratories) and reacted with 3′, 3-diaminobenzidine (Sigma-Aldrich). Sections were air-dried overnight and then dehydrated with graded alcohols, cleared in Histoclear and coverslipped with Permount (Fisher Scientific).

Image analysis.

Digital images from the apex of dentate gyrus where the dorsal an ventral horn meet, a contralateral region homologous to the depth electrode placement, were captured with an Olympus BX-61 microscope, using Olympus Microsuite software (Olympus America), with bright-field illumination at 10× magnification. A 300 μm² box was placed within the region of interest, and densitometric analysis was performed, under blinded conditions, in NIH ImageJ. Signal pixels of a region-of-interest were defined as having gray values 3.5 SD above the mean gray value of a cell-poor area close to the region of interest. The number of pixels and the average gray values above the set background were then computed for each region of interest and multiplied, giving an integrated densitometric measurement. Six measurements were made for each animal; the measurements were then averaged to obtain a single integrated density value per rat.

Statistics.

Results are expressed as mean ± SEM. A one-way repeated-measures ANOVA was used to compare the effect of stress (shock) on freezing behavior. A χ² test of goodness-of-fit was performed to determine whether nesting behavior was expressed equally between prenatally stressed dams compared with controls and t tests were used to analyze weight in dams (E3, E9, E15, and E21) and pups (P2), as well as litter size. All other analysis used a two-way ANOVA with stress and terbutaline as the independent factors. Fischer's LSD was used to conduct planned pairwise comparisons to follow-up significant overall ANOVAs. Data were analyzed using SPSS Statistics software and, in all cases, statistical significance was set at p < 0.05.