The influence of antibiotic treatment on the behavior and gut microbiome of adult rats neonatally insulted with lipopolysaccharide.

The present study investigated whether neonatal exposure to the proinflammatory endotoxin lipopolysaccharide (LPS) followed by an antibiotic (ATB)-induced dysbiosis in early adulthood could induce neurodevelopmental disorders-like behavioral changes in adult male rats. Combining these two stressors resulted in decreased weight gain, but no significant behavioral abnormalities were observed. LPS treatment resulted in adult rats' hypoactivity and induced anxiety-like behavior in the social recognition paradigm, but these behavioral changes were not exacerbated by ATB-induced gut dysbiosis. ATB treatment seriously disrupted the gut bacterial community, but dysbiosis did not affect locomotor activity, social recognition, and acoustic reactivity in adult rats. Fecal bacterial community analyses showed no differences between the LPS challenge exposed/unexposed rats, while the effect of ATB administration was decisive regardless of prior LPS exposure. ATB treatment resulted in significantly decreased bacterial diversity, suppression of Clostridiales and Bacteroidales, and increases in Lactobacillales, Enterobacteriales, and Burkholderiales. The persistent effect of LPS on some aspects of behavior suggests a long-term effect of early toxin exposure that was not observed in ATB-treated animals. However, an anti-inflammatory protective effect of ATB cannot be assumed because of the increased abundance of pro-inflammatory, potentially pathogenic bacteria (Proteus, Suttrella) and the elimination of the bacterial families Ruminococcaceae and Lachnospiraceae, which are generally considered beneficial for gut health.

Monitoring of kynurenine pathway metabolites, neurotransmitters and their metabolites in blood plasma and brain tissue of individuals with latent toxoplasmosis.

The aim of the presented work was to develop a highly sensitive, accurate and rapid analytical method for the determination of concentration levels of tryptophan and its metabolites of kynurenine catabolic pathway, as well as neurotransmitters and their metabolites in complex biological matrices (brain tissue and blood plasma). The developed analytical method consists of analytes separation from the biological matrices by protein precipitation (blood plasma) or solvent extraction (brain tissue), derivatization of the analytes and their detection by high-performance liquid chromatography combined with mass spectrometry. Individual steps of the whole process were optimized and the method was validated in the terms of selectivity, linearity (R2≥0.980), precision (RSD ≤ 13.3%), recovery (≥82.0%), limit of detection (1.8 ng/mL of blood plasma, 2.2 pg/mg of brain tissue) and limit of quantification (2.5 ng/mL of blood plasma, 2.8 pg/mg of brain tissue). The method was subsequently verified by an animal study, where the concentration levels of the analytes in biological matrices (blood plasma and brain tissue) of T. gondii - infected rats and control animals were compared. All the data obtained from the animal study were statistically evaluated. Increased concentration levels of kynurenine catabolic pathway metabolites (e.g. kynurenine, 3-hydroxykynurenine, quinolinic acid) were observed in the case of T. gondii - infected rats in contrast to the control group. The opposite effect was determined in the case of serotonin and its metabolite 5-hydroxyindoleacetic acid, where higher concentration levels were found in blood plasma of healthy subjects. Finally, Principal Component Analysis (PCA) was utilized for a score plot formation. PCA score plots have demonstrated the similarities of individuals within each group and the differences among the groups.

Neonatal immune activation by lipopolysaccharide causes inadequate emotional responses to novel situations but no changes in anxiety or cognitive behavior in Wistar rats.

Infection during the prenatal or neonatal stages of life is considered one of the major risk factors for the development of mental diseases such as schizophrenia or autism. However, the impacts of such an immune challenge on adult behavior are still not clear. In our study, we used a model of early postnatal immune activation by the application of bacterial endotoxin lipopolysaccharide (LPS) to rat pups at a dose of 2 mg/kg from postnatal day (PD) 5 to PD 9. In adulthood, the rats were tested in a battery of tasks probing various aspects of behavior: spontaneous activity (open field test), social behavior (social interactions and female bedding exploration), anxiety (elevated plus maze), cognition (active place avoidance in Carousel) and emotional response (ultrasonic vocalization recording). Moreover, we tested sensitivity to acute challenge with MK-801, a psychotomimetic drug. Our results show that the application of LPS led to increased self-grooming in the female bedding exploration test and inadequate emotional reactions in Carousel maze displayed by ultrasonic vocalizations. However, it did not have serious consequences on exploration, locomotion, social behavior or cognition. Furthermore, exposition to MK-801 did not trigger social or cognitive deficits in the LPS-treated rats. We conclude that the emotional domain is the most sensitive to the changes induced by neonatal immune activation in rats, including a disrupted response to novel and stressful situations in early adulthood (similar to that observed in human patients suffering from schizophrenia or autism), while other aspects of tested behavior remain unaffected.

Biochemical, histopathological and morphological profiling of a rat model of early immune stimulation: relation to psychopathology.

Perinatal immune challenge leads to neurodevelopmental dysfunction, permanent immune dysregulation and abnormal behaviour, which have been shown to have translational validity to findings in human neuropsychiatric disorders (e.g. schizophrenia, mood and anxiety disorders, autism, Parkinson's disease and Alzheimer's disease). The aim of this animal study was to elucidate the influence of early immune stimulation triggered by systemic postnatal lipopolysaccharide administration on biochemical, histopathological and morphological measures, which may be relevant to the neurobiology of human psychopathology. In the present study of adult male Wistar rats we examined the brain and plasma levels of monoamines (dopamine, serotonin), their metabolites, the levels of the main excitatory and inhibitory neurotransmitters glutamate and γ-aminobutyric acid and the levels of tryptophan and its metabolites from the kynurenine catabolic pathway. Further, we focused on histopathological and morphological markers related to pathogenesis of brain diseases--glial cell activation, neurodegeneration, hippocampal volume reduction and dopaminergic synthesis in the substantia nigra. Our results show that early immune stimulation in adult animals alters the levels of neurotransmitters and their metabolites, activates the kynurenine pathway of tryptophan metabolism and leads to astrogliosis, hippocampal volume reduction and a decrease of tyrosine hydroxylase immunoreactivity in the substantia nigra. These findings support the crucial pathophysiological role of early immune stimulation in the above mentioned neuropsychiatric disorders.

Neonatal administration of N-acetyl-L-aspartyl-L-glutamate induces early neurodegeneration in hippocampus and alters behaviour in young adult rats.

N-acetyl-L-aspartyl-L-glutamate (NAAG) is a dipeptide that could be considered a sequestered form of L-glutamate. As much as 25% of L-glutamate in brain may be present in the form of NAAG. NAAG is also one of the most abundant neuroactive small molecules in the CNS: it is an agonist at Group II metabotropic glutamate receptors (mGluR II) and, at higher concentrations, at the N-methyl-D-aspartate (NMDA) type of ionotropic glutamate receptors. As such, NAAG can be either neuroprotective or neurotoxic and, in fact, both characteristics have been discussed and described in the literature. In the present studies, 250 nmol NAAG was infused into each lateral cerebral ventricle of 12-day-old rat pups and, using Nissl-stained sections, neurodegeneration in the hippocampus was evaluated 24 or 96 h after the infusion. In several experiments, the neuronal death was also visualised by Fluoro-Jade B staining and studied by TUNEL technique. Some of the NAAG-treated animals were allowed to survive until 50 days post partum and subjected to behavioural (open field) tests. The administration of NAAG to 12-day-old rats resulted in extensive death of neurons particularly in the dentate gyrus of the hippocampus. The neurodegeneration was, in part, prevented by administration of an NMDA receptor antagonist MK-801 (0.1 mg/kg). The nuclear DNA-fragmentation demonstrated by TUNEL technique pointed to the presence of non-specific single-strand DNA cleavage. The NAAG-associated neonatal neuronal damage may have perturbed development of synaptic circuitry during adolescence as indicated by an altered performance of the experimental animals in the open field testing (changes in grooming activity) at postnatal day 50. The results underscore the potential neurotoxicity of NAAG in neonatal rat brain and implicate neonatally induced, NMDA receptor-mediated neuronal loss in the development of abnormal behaviour in young adult rats.

Simultaneous determination of dehydroepiandrosterone, its 7-hydroxylated metabolites, and their sulfates in rat brain tissues.

A method is described for simultaneous assessment of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and their 7-hydroxylated metabolites in cortex and subcortex of the rat brain. The procedure for determination of unconjugated steroids and DHEAS involved diethyl ether extraction of the homogenized tissue, solvent partition of the dry extract, and final quantification by specific radioimmunoassays. In addition, determination of 7-hydroxy-dehydroepiandrosterone sulfates required solvolysis, followed by high-performance liquid chromatography for separation of 7-hydroxylated metabolites from their precursor. The losses during this process were monitored by measurement of spiked radioactivity of [(3)H]testosterone or [(3)H]dehydroepiandrosterone sulfate. The content of dehydroepiandrosterone sulfate in both brain tissues was of the order of ten(s) nmol/g tissue irrespective its type (cortex or subcortex), while concentrations of other steroids were about 10 times lower in both tissues. In contrast to the ratio of sulfated/unconjugated DHEA, the levels of unconjugated 7-hydroxylated metabolites and their sulfates were close to each other. The reproducibility of the method with respect to coefficients of variation varied from 12 to 25%. An age-related decrease of sulfated dehydroepiandrosterone in the cortex of animals was also observed.