Prenatal Immune Challenge in Mice Leads to Partly Sex-Dependent Behavioral, Microglial, and Molecular Abnormalities Associated with Schizophrenia

Epidemiological studies revealed that environmental factors comprising prenatal infection are strongly linked to risk for later development of neuropsychiatric disorders such as schizophrenia. Considering strong sex differences in schizophrenia and its increased prevalence in males, we designed a methodological approach to investigate possible sex differences in pathophysiological mechanisms. Prenatal immune challenge was modeled by systemic administration of the viral mimic polyinosinic-polycytidylic acid (Poly I:C) to C57BL/6 mice at embryonic day 9.5. The consequences on behavior, gene expression, and microglia—brain immune cells that are critical for normal development—were characterized in male vs. female offspring at adulthood. The cerebral cortex, hippocampus, and cerebellum, regions where structural and functional alterations were mainly described in schizophrenia patients, were selected for cellular and molecular analyses. Confocal and electron microscopy revealed most pronounced differences in microglial distribution, arborization, cellular stress, and synaptic interactions in the hippocampus of male vs. female offspring exposed to Poly I:C. Sex differences in microglia were also measured under both steady-state and Poly I:C conditions. These microglial alterations were accompanied by behavioral impairment, affecting for instance sensorimotor gating, in males. Consistent with these results, increased expression of genes related to inflammation was measured in cerebral cortex and hippocampus of males challenged with Poly I:C. Overall, these findings suggest that schizophrenia's higher incidence in males might be associated, among other mechanisms, with an increased microglial reactivity to prenatal immune challenges, hence determining disease outcomes into adulthood.

performed on two separate days. All objects used were made from steel without odor and with protection against corrosion. The time for approaching each object was recorded by the ANYmaze system and the time of interaction with novel objects calculated. The open field platform was cleaned with 70% ethanol between animals.

Elevated plus maze
Elevated plus maze was performed to measure anxiety in a more sensitive manner. The apparatus for elevated plus maze consisted of a cross chamber (made of laminated cardboard), with two open arms and two closed arms surrounded by walls. Each arm measured 5cm x 30cm. The whole apparatus was elevated to 30cm. Each mouse was placed in the center of the chamber, with its head facing the upper closed arm, and was allowed to freely explore the arms for 10 min. For each tested animal, the number of arm entries, distance travelled, and time spent in the open and closed arms were recorded by the ANY-maze system. The elevated plus maze was cleaned with 70% ethanol between animals.

Three-chambered social interaction
Sociability and social novelty were measured with the three-chambered social interaction method of Crawley (Kaidanovich-Beilin et al., 2011). The apparatus consisted of a rectangular box in which each chamber measured 20cm x 40cm. Walls were made of transparent Plexiglas. Small open doors between the three chambers allowed for free circulation of the animals across chambers. Two identical, vertically placed, and cup-like containers, large enough to hold a single mouse, were used. The containers, one in each side chamber, served to enclose the intruder (unfamiliar mouse of the same strain, sex and age as the tested animal). During habituation, the animals were placed in the middle chamber and allowed to explore the area for 10 min. After cleaning with 70% ethanol, an intruder was placed in one of the containers to test for sociability.
The tested animal was allowed to search and interact with the intruder for 10 min. After cleaning the chamber with soap water, a second intruder was placed in the other container to assess social novelty and the tested animal was allowed to interact with both intruders for another 10 min. The interaction time with each intruder and total distance traveled of the tested animal were measured by the ANY-maze system.

RIKEN modified SHIRPA behavioral assessment
Modified SHIRPA (version 4) involved 68 tests used to evaluate morphology, behavior, sensory response, and athletic ability. The detailed version of the SHIRPA protocol and scoring methods are provided on the webpage of RIKEN BioResource Center: http://ja.brc.riken.jp/lab/jmc/shirpa/.
Briefly, mice were first evaluated under a transparent viewing jar made of Plexiglas during 5 min for body position, movement type, body position, respiration rate, body tremor, and the presence of fecal boli or urine. The mice were placed in the open field arena (55cm x 33cm) to test for transfer arousal, locomotor activity, piloerection, gait, auditory startle to a 90 db tone provided by a metal clicker, pelvic elevation, tail position, and touch escape. The mice were then lifted by the tail for 30 s and assessed for the position at which struggling movements occurred, as well as trunk curl and limb grasping. After landing on the horizontal grid, the mice were evaluated for grasping, grip strength when dragged by the tail, body tone after finger compression on each side, pinna and corneal reflexes, as well as toe-pinch withdrawal of a hindlimb upon squeezing with a hand-held forceps. After restraint in a supine position, body and tail lengths, eye conditions, skin color, heart rate, hindlimb tone, abdominal tone, lacrimation, salivation, and provoked biting in response to a plastic probe gently put in their mouth were observed. The forepaws of mice were then placed on a horizontal bar and their ability to hang suspended (wire maneuver) was assessed.
The drop righting reflex was evaluated according to landing position, followed by contact righting when placed inside of a small plastic tube. In the final phase, the mice were placed on a horizontal grid rotated towards a vertical position and assessed for negative geotaxis.
Neurological signs, such as abnormal reflexes, notably limb-clasping or a bat-like posture, and seizure activity, were recorded. These observations were completed by noting any signs of fear, irritability, aggression, vocalization, convulsion, strange behavior, and measuring body weight.

Prepulse inhibition
After SHIRPA, mice performed prepulse inhibition (PPI) of the acoustic startle response (ASR) from P60-P70, using an SR-LAB system (San Diego Instruments, San Diego, CA, USA). Tone pulse parameters were controlled by a microcomputer using the software package (SR-LAB) and interface assembly that also digitized (0-4095), rectified, and recorded stabilimeter readings. The procedures for PPI were modified from a previously published protocol (Kamath et al., 2008).
Animals were placed in a Plexiglas enclosure and allowed to acclimatize to their environment with a background noise of 70 dB for 5 min before being tested during 42 discrete trials. On the first two trials, the magnitude of the startle response to a 120 dB tone of 30 ms duration was measured. These first two startle tones were presented to habituate the animals to the testing procedure and thus were omitted from the data analysis. On the subsequent 40 trials, the startle tone was either presented alone or 100 ms after presentation of prepulses of 30 ms duration with intensities ranging from 0 dB to 15 dB above background noise (i.e. 70-85 dB) that varied randomly between the trials. The prepulse-pulse trials at 70dB were presented to compare with startle response at 120 dB only and were omitted from the analysis. ASR was measured at each of the six prepulse intensities over five trials; animals were randomly presented with the startle tone alone during the other 10 trials. The interval between each trial was programmed to a variable schedule with an average duration of 15 s (range 5-25 s). A measure of ASR amplitude was derived from the mean of 100 digitized data-points collected from stimulus onset at a rate of 1 kHz. Startle response was recorded for each pulse-alone and prepulse+pulse trial. Startle magnitude was calculated as the average of the startle responses to the pulse-alone trials. The prepulse effectiveness at suppressing ASR was expressed as a percentage based on the mean amplitude of responses to ten startle tones alone relative to those recorded under the five prepulse conditions (n=5 per condition) where: %PPI= 100-(startle response for prepulse + pulse trial x 100% / startle response for pulse-alone trial).

Supporting Figure Legends
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