Neonatal behavioral changes in rats with gestational exposure to lipopolysaccharide: a prenatal infection model for developmental neuropsychiatric disorders

Abstract

Exposure to prenatal infections has been widely associated with the increased risk for neuropsychiatric disorders of developmental origin such as schizophrenia and autism. Although several behavioral and cognitive deficits have been detected during adulthood in rodent models of prenatal infections, early behavioral changes have not been well characterized. In a prenatal lipopolysaccharide (LPS) model, we have previously observed significant alterations in the neuronal cytoarchitecture during early postnatal life. In the present study, we aimed to investigate the potential effects of prenatal immune activation on early neurophenotypic presentations using a set of behavioral test battery. Female Sprague-Dawley rats were administered with 100 μg/kg LPS (intraperitoneally) at gestational days 15 and 16. During the first postnatal week, we found no significant effect on maternal behavior or mother-pup interaction by this treatment. Also, no major changes in physical developmental milestones of pups were noted from postnatal (P) days P6 to P16. Importantly, prenatal LPS-exposed pups had a significant decrease in the number and duration of ultrasonic vocalization calls at P3 and P5. Prenatal LPS treatment also led to impairments in nest-seeking behavior and odor-stroke associative learning in neonatal rats at P8 and P9. At the molecular level, we detected significant decrease in the expression of cortical 5HT1A and 5HT1B messenger RNA at P3. These data suggest that prenatal exposure to an immune activator can significantly impair the social/communicative behavior in the neonate offspring, which may be relevant to childhood and premorbid abnormalities reported in autism and schizophrenia subjects.

Fig. 1.

Effect of Prenatal Lipopolysaccharide (LPS) Treatment on Maternal Behavior at Days P4–P5 During Light Phase (Left Panel) and Dark Phase (Right Panel) (10–12 Dams Per Treatment). (A, B) Frequency of active nursing, passive nursing, and grooming of the pups were calculated over a period of 72 min in observation bins of 3 min. (C, D) Active nursing was further subdivided into 2 categories: high and low arch back nursing and frequency of behaviors was measured for LPS- and saline-treated dams. (E, F) Frequency of dam-related behaviors such as active wandering, passive wandering, and eating/drinking or self-grooming were also compared between 2 prenatal treatment conditions. (G, H) Nest building. Values are presented as means ± standard error of the mean.

Fig. 2.

Physical Developmental Milestones and Reflex Development in Pups Exposed to 100 μg/kg Lipopolysaccharide (LPS) or Saline at Gestational Age 15–16 (Mean of 6 Litters Per Treatment). (A) Weight gain: assessed at birth and during days P6–P16; (B, C) time of eye and ear canal opening: monitored from days P13 to P17; and (D) forelimb grasp reflex: the reflex was delayed for 1 d in LPS-treated pups comparing to saline controls (*P < .05). In (B–D), data shown as percentage of pups per litter fully performed the task. Values are expressed as mean ± standard error of the mean. (*P < .05 LPS- vs saline-treated offspring).

Fig. 3.

Physical Developmental Milestones and Reflex Development in Pups Exposed to 100 μg/kg Lipopolysaccharide (LPS) or Saline at Gestational Age 15–16 (Mean of 6 Litters Per Treatment). (A) Grip strength response (I): data presented based on the percentage of pups in each litter that could perform the task; (B) Grip strength response (II): data presented as the time required for each pup to hold the metal bar. LPS-treated pups could suspend themselves for significantly longer time comparing to saline (*P < .05); (C) Righting reflex: based on the time required for each pup to return to its four limbs when it is placed on its back and the mean values were determined for each litter; (D, E) Geotactic reaction and auditory startle: data shown as percentage of pups per litter fully performed the task; and (F) Cliff avoidance: based on the time required for each pup to retract from the edge of a flat surface itself and the mean values were determined for each litter. All values are expressed as mean ± standard error of the mean (*P < .05 LPS- vs saline-treated offspring).

Fig. 4.

(A) Locomotor activity (n = 24–27 per treatment): the scoring was done based on the number of line crossings by each pup during 3-minute observation period. The data were analyzed using 2-way ANOVA with time as repeated measurement. (B) Nest-seeking behavior (n = 16–20 per treatment): data presented as number of approaches toward maternal nest (observation time: 60 s). Each pup was assigned to 2 trials. The data were analyzed by 2-tailed Student t test (*P < .05). (C) Nest exploration: number of choices toward maternal nest plus exploration of the nest was considered a positive score and analyzed by 2-tailed Student t test (P = .0692). (D) Odor-stroke associative learning: number of choices toward the conditioned odor at day P9 in saline- and lipopolysaccharide (LPS)-treated offspring that received either paired odor-stroke (n = 10–11) or unpaired odor-stroke (n = 8–9) or odor-only (n = 8–10) presentation on the training day (P8). Two-way ANOVA of data showed that in saline group, odor-stroke paired pups made more entries toward the side of conditioned odor compared with unpaired or odor-only groups (*P < .05 and **P < .01, respectively). Furthermore, within paired group, the LPS-treated pups made significantly fewer choices toward conditioned odor comparing to saline (#P < .05).

Fig. 5.

Isolation-Induced Ultrasonic Vocalization Calls (USVs) During 3-min Observation Time (n = 12–18 Per Treatment). (A) Number of USV calls of the pups isolated from their mother and littermates at days P3, P5, P9, and P11. Number of USVs was significantly reduced in lipopolysaccharide (LPS)-treated pups at day P3 (*P < .05). (B) Total duration of USV calls for each pup at days P3, P5, P9, and P11. Total duration of calls was significantly decreased in LPS-treated offspring at day P5 (***P < .001). (C) Mean duration of USVs for each pup at days P3, P5, P9, and P11. The mean duration of each was also reduced in LPS-treated offspring at day P5 (***P < .001). (D) Total number of USV calls during first isolation and second isolation right after brief maternal contact in prenatal saline- and LPS-treated offspring at day P9 (n = 12 per treatment). All the data are presented as mean ± standard error of the mean (*P < .05 and ***P < .001).