Embryonic exposure to valproic acid (VPA) is known to produce sociability deficits, resembling human autistic phenotypes, in several vertebrate species. Animals living in groups prefer the proximity of peers and have the ability to perceive and to respond to social signals for modifying behavior. Chicks of Galliform birds, known to display early preference behaviors, have been used extensively for adaptive learning studies. Young precocial birds seem to be useful models also for studying the effect of embryonic VPA treatment. Here, domestic chicken eggs were injected with sodium valproate (200 μl of 35 μmol/L solution) or with vehicle (distilled water) on the 14th day of incubation. After hatching, the chicks were tested for one-trial passive avoidance learning at day 1, vocalization due to isolation as a measure of stress level (day 2), approach preference to large versus small groups of age-matched conspecifics (day 5), and to those with normal versus blurred head features (day 7). In addition, we tested the preference of birds to conspecifics reared in group versus those reared in isolation (day 9), as well as the preference of chicks to familiar versus non-familiar conspecifics (day 21). Our findings confirm previous reports concerning an adverse effect of VPA on embryonic development, including a tendency for aborted or delayed hatching and, occasionally, for locomotor disorders in a small percentage of birds (eliminated from later studies). Otherwise, VPA treatment did not impair motor activity or distress level. Memory formation for the aversive stimulus and discrimination of colors were not impaired by VPA treatment either. Innate social predispositions manifested in approach preferences for the larger target group or for the birds with natural facial features remained unaffected by VPA exposure. The most prominent finding was attenuation of social exploration in VPA-exposed birds (expressed as the frequency of positional switches between two stimulus chicks after the first choice), followed by a deficit in the recognition of familiar conspecifics, unfolding at the end of the third week. Social exploration and recognition of familiar individuals are the key elements impaired at this stage. The results underline the importance of early social exploration in ASD.

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Behaviour of young domestic chicks when isolated from conspecifics is influenced by two conflicting drives: fear of potential predator and craving for company. The nucleus accumbens (Ac) has been suggested to influence social behaviour, as well as motivation in goal-directed tasks. In this study, the Ac of 1-day-old domestic chicks was lesioned bilaterally, using radiofrequency method. Open field behaviour before and after presenting a silhouette of a bird of prey was recorded, followed by a behavioural test to measure group size preference and social motivation of chicks. Ac-lesioned individuals emitted more distress calls and ambulated more in the open field test, however, they reacted to the predatory stimulus very similarly to control chicks: their vocalization was reduced and the intergroup difference in motor activity also disappeared. There was no difference between the lesioned and control chicks in the latency to approach their conspecifics in the social motivation test, and both groups chose the larger flock (eight) of conspecifics over the smaller one (three). Concerning the role of Ac in social behaviour, a difference between lesioned and sham birds was evident here only in the absence of detectable stimulus (predator or conspecifics). These findings may reflect either decreased fear of exposure to predators or increased craving for conspecifics suggesting that the likely function of Ac is to modulate goal-driven, including socially driven, behaviours, especially when the direct stimulus representing the goal is absent. This is in harmony with the known promotion of impulsiveness by Ac lesions.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) induce significant damage to the small intestine, which is accompanied by changes in intestinal bacteria (dysbiosis) and bile acids. However, it is still a question of debate whether besides mucosal inflammation also other factors, such as direct antibacterial effects or delayed peristalsis, contribute to NSAID-induced dysbiosis. Here we aimed to assess whether ketorolac, an NSAID lacking direct effects on gut bacteria, has any significant impact on intestinal microbiota and bile acids in the absence of mucosal inflammation. We also addressed the possibility that ketorolac-induced bacterial and bile acid alterations are due to a delay in gastrointestinal (GI) transit.Methods: Vehicle or ketorolac (1, 3 and 10 mg/kg) were given to rats by oral gavage once daily for four weeks, and the severity of mucosal inflammation was evaluated macroscopically, histologically, and by measuring the levels of inflammatory proteins and claudin-1 in the distal jejunal tissue. The luminal amount of bile acids was measured by liquid chromatography-tandem mass spectrometry, whereas the composition of microbiota by sequencing of bacterial 16S rRNA. GI transit was assessed by the charcoal meal method.Results: Ketorolac up to 3 mg/kg did not cause any signs of mucosal damage to the small intestine. However, 3 mg/kg of ketorolac induced dysbiosis, which was characterized by a loss of families belonging to Firmicutes (Paenibacillaceae, Clostridiales Family XIII, Christensenellaceae) and bloom of Enterobacteriaceae. Ketorolac also changed the composition of small intestinal bile by decreasing the concentration of conjugated bile acids and by increasing the amount of hyodeoxycholic acid (HDCA). The level of conjugated bile acids correlated negatively with the abundance of Erysipelotrichaceae, Ruminococcaceae, Clostridiaceae 1, Muribaculaceae, Bacteroidaceae, Burkholderiaceae and Bifidobacteriaceae. Ketorolac, under the present experimental conditions, did not change the GI transit.Conclusion: This is the first demonstration that low-dose ketorolac disturbed the delicate balance between small intestinal bacteria and bile acids, despite having no significant effect on intestinal mucosal integrity and peristalsis. Other, yet unidentified, factors may contribute to ketorolac-induced dysbiosis and bile dysmetabolism.