Erika Roman

Behavioral profiles and stress-induced corticosteroid secretion in male Wistar rats subjected to short and prolonged periods of maternal separation

Early life experiences are important for the development of neurobiobehavioral mechanisms and subsequent establishment of mental functions. In experimental animals, early life experiences can be studied using the maternal separation model. Maternal separation has been described to induce neurobiological changes and thus affect brain function, mental state and behavior. We have established a protocol in order to study the effects of repeated short and prolonged periods of maternal separation during the postnatal period on adult neurochemistry, voluntary ethanol intake and behavior. In the present experiment, we focus on the long-term effects of maternal separation on exploration and risk assessment behavior as well corticosteroid secretion. Rat pups were assigned to 15 min (MS15) or 360 min (MS360) of daily maternal separation and normal animal facility rearing (AFR) during postnatal days 1-21. To establish the adult behavioral profile in male rats, three tests were used: the Concentric Square Field (CSF), the Open Field (OF) and the Elevated Plus-maze (EPM). No differences between the three experimental groups were found in the traditional OF and EPM tests. The CSF test indicated that the MS360 rats were more explorative and expressed an altered risk assessment and risk-taking profile. In response to a restraint stress, MS360 rats had a blunted corticosterone release in contrast to MS15 and AFR rats. In contrast to previous results, the outcome of the present investigation does not support the notion that a prolonged period of maternal separation results in an adult phenotype characterized by an increased emotional reactivity.

The impact of emotional stress early in life on adult voluntary ethanol intake-results of maternal separation in rats

The combination of genetic and environmental factors determines the individual vulnerability for excessive ethanol intake, possibly leading to dependence. The environmental influences early in life represent examples of determinant factors for adult behaviour and can be protective as well as risk factors. Maternal separation is one model to examine the long-term consequences of early environmental experiences on neurochemistry and behaviour, including drug-taking behaviour in experimental animals. In the present review, findings from studies using repeated short and prolonged periods of maternal separation, with emphasis on effects on voluntary ethanol intake in rats with or without a genetic predisposition for high voluntary ethanol intake, are summarized. Despite some contradictory results, the general picture emerging shows that short periods of maternal separation during the postnatal period result in a lower adult voluntary ethanol intake in male rats. Prolonged periods of maternal separation were found to induce a high voluntary ethanol intake in male rats, including rats with a genetic predisposition for high ethanol intake. Results from the literature also show that changes were not just related to time of separation but were also related to the degree of handling. Interestingly, in terms of voluntary ethanol intake, female rats were generally not affected by postnatal maternal separation. The reasons for these sex differences need further investigation. In terms of neurobiological consequences of maternal separation, conclusive data are sparse and one of the future challenges will, therefore, be to identify and characterize underlying neurobiological mechanisms, especially in the individual animal.

Selective Brain Uptake and Behavioral Effects of the Cyanobacterial Toxin BMAA (β-N-Methylamino-L-alanine) following Neonatal Administration to Rodents

Cyanobacteria are extensively distributed in terrestrial and aquatic environments all over the world. Most cyanobacteria can produce the neurotoxin beta-N-methylamino-L-alanine (BMAA), which has been detected in several water systems and could accumulate in food chains. The aim of the study was to investigate the transfer of BMAA to fetal and neonatal brains and the effects of BMAA on the development of behavioral characteristics during the brain growth spurt (BGS) in rodents. Pregnant and neonatal mice were given an injection of (3)H-BMAA on gestational day 14 and postnatal day (PND) 10, respectively, and processed for tape-section autoradiography. The study revealed transplacental transfer of (3)H-BMAA and a significant uptake in fetal mouse brain. The radioactivity was specifically located in the hippocampus, striatum, brainstem, spinal cord and cerebellum of 10-day-old mice. The effect of repeated BMAA treatment (200 or 600 mg/kg s.c.) during BGS on rat behavior was also studied. BMAA treatment on PND 9-10 induced acute alterations, such as impaired locomotor ability and hyperactivity, in the behavior of neonatal rats. Furthermore, rats given the high dose of BMAA failed to habituate to the test environment when tested at juvenile age. In conclusion, the results demonstrated that BMAA was transferred to the neonatal brain and induced significant changes in the behavior of neonatal rats following administration during BGS. The observed behavioral changes suggest possible cognitive impairment. Increased information on the long-term effects of BMAA on cognitive function following fetal and neonatal exposure is required for assessment of the risk to childrens health.

Long-term effects of short and long periods of maternal separation on brain opioid peptide levels in male Wistar rats.

Environmental manipulations early in life may induce persistent alterations in adult behaviour and physiology. The underlying neural mechanisms of these responses are not yet clear. We have previously reported long-term changes in brain opioid peptide levels in male and female Sprague-Dawley rats after short periods (15 min, known as neonatal handling) of maternal separation (MS) until weaning. To study this further, we investigated behavioural and neurochemical effects of repeated MS in male Wistar rats. The rat pups were separated from their dams in litters for either 360 min (MS360) or 15 min (MS15) daily from postnatal day 1 to 21 or exposed to normal animal facility rearing. Behavioural analysis showed that MS360 rats had increased ultrasonic calls on postnatal day 5 compared to MS15 rats, but not on postnatal day 6. Moreover, the MS360 rats had more animals with higher frequency of calls at day 5 than 6 than the MS15 rats. Analysis of the opioid peptides dynorphin B and Met-enkephalin-Arg(6)Phe(7) with radioimmunoassay 7 weeks after the MS procedure, revealed long-term neurochemical changes in several brain areas and in the pituitary gland. Immunoreactive dynorphin B and Met-enkephalin-Arg(6)Phe(7) levels were affected in the hypothalamus and dynorphin B levels in the neurointermediate pituitary lobe, amygdala, substantia nigra and the periaqueductal gray. Together, these findings show that repeated periods of MS early in life in male Wistar rats affect the development of the ultrasonic call response and induce long-lasting and possibly permanent alterations in the opioid peptide systems.

Neonatal Exposure to the Cyanobacterial Toxin BMAA Induces Changes in Protein Expression and Neurodegeneration in Adult Hippocampus

The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) has been proposed to contribute to neurodegenerative disease. We have previously reported a selective uptake of BMAA in the mouse neonatal hippocampus and that exposure during the neonatal period causes learning and memory impairments in adult rats. The aim of this study was to characterize effects in the brain of 6-month-old rats treated neonatally (postnatal days 9–10) with the glutamatergic BMAA. Protein changes were examined using the novel technique Matrix-Assisted Laser Desorption Ionization (MALDI) imaging mass spectrometry (IMS) for direct imaging of proteins in brain cryosections, and histological changes were examined using immunohistochemistry and histopathology. The results showed long-term changes including a decreased expression of proteins involved in energy metabolism and intracellular signaling in the adult hippocampus at a dose (150mg/kg) that gave no histopathological lesions in this brain region. Developmental exposure to a higher dose (460mg/kg) also induced changes in the expression of S100β, histones, calcium- and calmodulin-binding proteins, and guanine nucleotide-binding proteins. At this dose, severe lesions in the adult hippocampus including neuronal degeneration, cell loss, calcium deposits, and astrogliosis were evident. The data demonstrate subtle, sometimes dose-dependent, but permanent effects of a lower neonatal dose of BMAA in the adult hippocampus suggesting that BMAA could potentially disturb many processes during the development. The detection of BMAA in seafood stresses the importance of evaluating the magnitude of human exposure to this neurotoxin.

Basal levels and alcohol-induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice.

In order to investigate the involvement of the opioid and nociceptin/orphanin FQ (N/OFQ) system in alcohol drinking behaviour, N/OFQ and the opioid peptides dynorphin B (DYNB) and Met-enkephalin-Arg(6) Phe(7) (MEAP) were examined in the alcohol-preferring C57BL/6J mice. Basal peptide levels were compared in the brain and the pituitary gland with basal levels in the alcohol-avoiding DBA/2J mice. Furthermore, the effects of chronic alcohol self-administration on peptides were studied in the C57BL/6J mice. Compared to the DBA/2J mice, C57BL/6J mice had low immunoreactive (ir) levels of DYNB and MEAP in the nucleus accumbens, the hippocampus, and the substantia nigra, low ir-DYNB levels in the striatum and low ir-MEAP levels in the frontal cortex. Higher ir-DYNB levels in the pituitary gland and in the periaqueductal gray (PAG) and higher ir-N/OFQ levels in the frontal cortex and in the hippocampus were detected in C57BL/6J mice compared to the DBA/2J mice. After 4 weeks of voluntary alcohol consumption, only minor changes in steady-state peptide levels were identified. However, 5 days after the alcohol-drinking period, lower levels of all peptides were detected in the ventral tegmental area and ir-DYNB levels were also lower in the amygdala and in the substantia nigra. Twenty-one days after cessation of alcohol self-administration, the opioid peptides in alcohol-consuming C57BL/6J mice were lower in the PAG, the N/OFQ was lower in the frontal cortex and DYNB was higher in the amygdala and substantia nigra as compared to control C57BL/6J mice. This study demonstrates strain differences between C57BL/6J mice and DBA/2J mice that could contribute to divergent drug-taking behaviour, and it also demonstrates time- and structure-specific changes in neuropeptide levels after alcohol self-administration in the C57BL/6J mice.

The Evolutionary History and Tissue Mapping of Amino Acid Transporters Belonging to Solute Carrier Families SLC32, SLC36, and SLC38

Members of the solute carrier families (SLC) 32, 36, and 38, together also designated the β-group of SLCs, are known to transport neutral amino acids. In this paper, we show that these three families were present before the split of the animal lineage and that they are likely to share a common decent. We also show that the APF transporters found in plants are most likely homologous to the mammalian β-group, suggesting that this type of transporters arouse early in the evolution of eukaryotes. We performed detailed tissue expression analysis of all the members of the β-group in rat and found several examples of highly specific expression patterns, with SLC38A7 being exclusively found in liver, SLC38A5 in blood, and SLC38A4 in muscle and liver. Moreover, we found that SLC38A10 is expressed in several endocrine organs. We also found that SLC38A1 is highly up regulated in the cortex from rats treated with diazepam and that SLC38A2 is significantly down regulated in the same tissue. In addition, we performed a detailed expression analysis of SLC38A1 and SLC38A6 in mouse brain using in situ hybridization, which showed that both these transporters are widely expressed in the brain.

Is the rodent maternal separation model a valid and effective model for studies on the early-life impact on ethanol consumption?

RationaleEarly-life events can cause long-term neurobiological and behavioural changes with a resultant effect upon reward and addiction processes that enhance risk to develop alcohol use disorders. Maternal separation (MS) is used to study the mediating mechanisms of early-life influences in rodents. In MS studies, the pups are exposed to maternal absence during the first postnatal weeks. The outcome of MS experiments exhibits considerable variation and questions have been raised about the validity of MS models.ObjectivesThis short review aims to provide information about experimental conditions that are important to consider when assessing the impact of early-life environment on voluntary ethanol consumption.ResultsThe results from currently used MS protocols are not uniform. However, studies consistently show that longer separations of intact litters predispose for higher ethanol consumption and/or preference in adult male rats as compared to shorter periods of MS. Studies using individual pup MS paradigms, other controls, low ethanol concentrations, adult females or examining adolescent consumption reported no differences or inconsistent results.ConclusionsThere is no “a rodent MS model”, there are several models and they generate different results. The compiled literature shows that MS is a model of choice for analysis of early-life effects on voluntary ethanol consumption but there are examples of MS paradigms that are not suitable. These studies emphasize the importance to carefully designed MS experiments to supply the optimal conditions to definitely test the research hypothesis and to be particulate in the interpretation of the outcome.

Early hippocampal cell death, and late learning and memory deficits in rats exposed to the environmental toxin BMAA (β-N-methylamino-l-alanine) during the neonatal period

We have reported previously that exposure to the cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) during the neonatal period causes cognitive impairments in adult rats. The aim of this study was to investigate the long-term effects of neonatal BMAA exposure on learning and memory mechanisms and to identify early morphological changes in the neonatal brain. BMAA was injected subcutaneously in rat pups on postnatal days 9-10. BMAA (50 and 200 mg/kg) caused distinct deficits in spatial learning and memory in adult animals but no morphological changes. No impairment of recognition memory was detected, suggesting that neonatal exposure to BMAA preferentially affects neuronal systems that are important for spatial tasks. Histopathological examination revealed early neuronal cell death as determined by TUNEL staining in the hippocampus 24 h after a high dose (600 mg/kg) of BMAA whereas no changes were observed at lower doses (50 and 200 mg/kg). In addition, there was a low degree of neuronal cell death in the retrosplenial and cingulate cortices, areas that are also important for cognitive function. Taken together, these results indicate that BMAA is a developmental neurotoxin inducing long-term changes in cognitive function. The risk posed by BMAA as a potential human neurotoxin merits further consideration, particularly if the proposed biomagnifications in the food chain are confirmed.

The Concentric Square Field: a multivariate test arena for analysis of explorative strategies.

In this study, we describe the behavior of laboratory rats in a recently developed observation arena, the Concentric Square Field (CSF). The CSF contains a number of areas designed to provoke exploration and behaviors associated with risk assessment, risk taking and security seeking in an environment not previously experienced. The model includes sheltered, open and elevated areas, a hole board device, areas with different light conditions, and wall-enclosed corridors. The rationale behind the CSF is to meet the demand for multivariate test situations that are not predictive in the sense of previous definition of a specific purpose of measuring a certain mental state. We define multivariate as being a free choice of where to stay in areas of different qualities. In the present study, identification of risky as opposed to safe areas is based on the retrieval behavior in lactating females and hoarding of food pellets in food-deprived males. Furthermore, we describe the effects of pre-trial food deprivation, immobilization, social stress, strain differences (Sprague-Dawley, Wistar and Lister Hooded males), sex differences (Sprague-Dawley) and repeated testing. Besides the conventional statistics, a principal component analysis (PCA) helped to discriminate between the various categories tested. Our conclusion is that the multivariate and non-predictive test situation (CSF) and the use of PCA provide a good tool for ethoexperimental analysis.