Approximately 10 years ago, “bath salts” were popularized as legal alternatives to the psychostimulants cocaine and the amphetamines, circumventing legislation with packages marked, “not for human consumption.” These products contained synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). The synthetic cathinones have similar pharmacology to controlled psychostimulants, increasing levels of dopamine (DA) in the synaptic cleft, while also exhibiting similar psychoactive effects such as increased energy and euphoria. Additionally, adverse effects of “bath salts” are similar to controlled psychostimulants, such as chest pain, shortness of breath, and hallucinations. Most preclinical investigations have only assessed the effects of these synthetic cathinones independently; however, case reports and DEA studies indicate that “bath salts” contain mixtures of these substances. Therefore, in a recent study by our laboratory, we examined effects of individual versus combined exposure to MDPV, mephedrone, and methylone. Interestingly, an enhanced effect on the levels of DA in a number of brain regions was observed, as well as significant alterations in locomotor activity following co-exposure to the cathinones. Here, we examine if the enhanced effects of the drug combination are due to pharmacokinetic (PK) interactions. Many of the same cytochrome isoenzymes metabolize each of these 3 drugs; thus, it is probable that the drugs’ PK would differ when administered individually as compared to in combination. We hypothesized that combined exposure to MDPV, mephedrone, and methylone would result in increased total drug concentrations when compared to individual administration. Briefly, adolescent male Swiss-Webster mice were injected intraperitoneally with either 10 mg/kg MDPV, 10 mg/kg mephedrone, 10 mg/kg methylone, or 10 mg/kg combined MDPV, mephedrone, and methylone. Following injection, brains and plasma were collected at the following time points: 1, 10, 15, 30, 60, and 120 minutes. Drugs were extracted via solid-phase extraction, and concentrations were determined using a previously published high pressure-liquid chromatography tandem mass spectrometry method. All drugs quickly crossed the blood-brain barrier and entered the brain. PK data for methylone and mephedrone was consistent with our hypothesis. For methylone, the maximal concentration (Cmax) and the total drug exposure (as represented by the area under the curve (AUC)) were significantly higher when combined with mephedrone and MDPV in both matrices. For mephedrone, the Cmax was unchanged, but AUC in brain was increased when combined with the other two drugs. However, interestingly, for MDPV, the Cmax was unchanged, yet the AUC in brain was higher when MDPV was administered individually. These data provide insight into the consequences of co-exposure to synthetic cathinones in popular “bath salt” products.

No description available.

O transcrito regulado pela cocaína e anfetamina (CART) é amplamente expresso em todo o sistema nervoso central. Ele está envolvido na manutenção de diversas funções neurofisiológicas como comportamento alimentar, comportamento sexual, memória e aprendizagem, controle do estresse, processos sensoriais e plasticidade neuronal. Evidências sugerem que o CART deve participar de processos relacionados ao desenvolvimento neural. Diante disso estudamos a expressão do CART em diferentes idades pós-natais com o intuito de identificar as possíveis regiões prosencefálicas nas quais este peptídeo pode estar participando do desenvolvimento neuronal. Por meio da técnica de hibridização in situ, comparamos a expressão do CART no prosencéfalo de animais de 6, de 26 e de 66 dias de vida. Observamos que os animais de 6 dias expressam uma maior quantidade de CART em várias regiões prosencefálicas comparado com animais jovens (26 dias) e adultos (66 dias). Estas áreas são: o córtex somatossensorial, o córtex piriforme, o indusim griseum, o giro denteado, o núcleo accumbens e o núcleo pré-mamilar ventral. Observamos também que o CART é expresso nos núcleos ventro-póstero lateral e ventro-póstero medial do tálamo nos animais de 6 dias, expressão que não é observada no animal adulto. Em outras regiões como o CA1 e o núcleo arqueado não observamos diferença. Utilizando imunohistoquímica, observamos que o peptídeo CART está presente nas regiões descritas. Nossos dados sugerem que o CART participa de processos relacionados ao desenvolvimento em diversas regiões do prosencéfalo. / Cocaine and amphetamine-regulated transcript (CART) is found widespread in the rodent nervous system. It has been involved in many different functions including feeding and sexual behaviors, learning and memory, stress responses, sensory processing and neuronal plasticity. Recent studies suggest that CART may be also implicated in neural development. Therefore, in the present study we investigated CART expression in the forebrain of rats in different stages of development. Using in situ hybridization, we compared CART expression in the forebrain of 6, 26 and 66 days old Sprague-Dawley rats. In general, we found a higher expression of CART in 6 days old animals comparing to other groups. Specifically, we found increased CART expression in the somatosensory and piriforme cortex, induzium griseum, dentate gyrus, nucleus accumbens, and ventral prepamammillary nucleus. Interestingly, we found CART expression in the ventral posteromedial and ventral posterolateral thalamic nuclei only in 6-days old animals. In other regions including the CA1 and the arcuate nucleus, CART expression did not change. By using immunohistochemistry we found that the CART peptide is produced in the areas where we found the mRNA, in 6 days old animals. Our findings suggest that the CART participate in neural development in various forebrain nuclei.

Abstract not available

The neuropeptides galanin and neuropeptide Y (NPY) play an important role in the reproduction of rodents, e.g. by modulating the release of gonadal hormones, the nutritional status by effects on feeding behavior and also by influencing mating behavior. There are age- and gender- differences in galanin- and NPY- like immunoreactivities (LIs) in brain areas important for higher functions including the hippocampal formation (HiFo) and cortex, that are related to the concentrations of 17β-estradiol. Neuropeptides in general are currently not considered critical in normal integrative neuronal functions but are rather thought to act as slow modulators during periods of stress or injury. In the present thesis we attempted to investigate, if the normal cyclical changes in the female sex-hormone 17β-estradiol can affect neurotransmission in brain areas important for memory, cognition and mood. We studied not only ”long term” (days and weeks) but also ”short-term” (one hour) effects on galanin and NPY concentrations in 17β-estradiol-primed ovariectomized (ovx) rats and mice. Radioimmunoassay (RIA) of galanin-LI in extracts of brain tissues from ”long-term” 17β-estradiol-treated ovx rats showed that its effects on galanin are dependent on boththe dose and on duration. Galanin - and NPY-LI in brain tissues of young ovx rats and mice increased in response to 17β-estradiol treatment in the HiFo, frontal cortex and striatum already within hours. This effect was not blocked by Tamoxifen® in rats. The mechanism of the 17β-estradiol effects on galanin levels in the rat HiFo may be related to decreased release of galanin into the extracellular fluid, since galanin-LI decreased in microdialysis samples two hours after a single injection of 17β-estradiol. Species differences were observed with regards to galanin, possibly due to tissue and species differences in the distribution of estrogen receptors. In the HiFo and caudate nucleus of mice, we found an increase in NPY-transcript after two hours by means of insitu hybridization, perhaps a compensatory up-regulation of NPY mRNA after increased 17β-estradiol-induced release in these areas. Taken together with no effects of Tamoxifen® on the levels on galanin in the HiFo of rats, the short duration, and the fact that the density of classical estrogen receptors seems to be limited in the striatum, we suggest that these effects are mediated through a membrane-related mechanism perhaps not involving the classical ER route. With an antiserum raised against the C-terminal end of the first 16 aminoacids of galanin- the sequence important for binding of intact galanin to its receptor - we found a novel compound which appears to be a homologue to galanin. Chromatographical analysis revealed that it was not galanin(1-29) or the galanin related peptide, galaninlike peptide (GALP), but appeared with immunohistochemistry in the galanin systems in the brain and was further influenced by 17β-estradiol in the HiFo and frontal cortex in a similar manner as galanin(1-29). In conclusion, tissue concentrations of galanin, a putative galanin homologue and NPY can be altered already after one hour by 17β-estradiol treatment e.i. in the HiFo. These ”short-term” effects are most likely to be due to effects on estrogen-primed peptide release which might influence mechanisms important for memory, cognition and mood.

The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows: i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status. ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo. iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated. iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity. v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites. vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

No description available.

Abnormal social behavior is a symptom of many human conditions, including Fetal Alcohol Spectrum Disorders (FASD). The zebrafish may be an excellent model to study embryonic alcohol effects, owing to the ease of drug administration. In recent decades, zebrafish have become increasingly popular in behavioral neuroscience, and their elaborate behavioral repertoire can provide insights in brain functioning and social behavior in response to teratogens or pharmaceutical agents. Shoaling is the zebrafish’s most notable behavioral feature, but has not been well characterized. I have started this characterization by describing the ontogeny of shoaling behavior. Embryonic exposure to low doses of ethanol has been shown to impair social behavior without any gross morphological alterations in zebrafish. However, this has not been studied in freely moving groups (shoals). Validation for using a shoaling task in behavioral testing, and potentially for high throughput analyses in the future, hinges on thorough characterization of the behavioral effects, however subtle in alcohol treated zebrafish. In my studies, I have shown that social behavior is impaired in zebrafish in a group setting after one time embryonic ethanol exposure. I have also discovered that this impairment is accompanied by a reduction of dopamine, serotonin, DOPAC and 5HIAA, neurochemicals measured in whole brain samples using HPLC. I have developed refined methods of measuring shoaling behavior in ten member zebrafish groups, laying the foundation for high throughput screening of adult zebrafish. Zebrafish are an economical model, and therefore lend themselves particularly well for high throughput screening. However, current paradigms are still labor intensive and require substantial human capital. By refining current behavioral tests and deploying new analytical tools, high throughput screening is starting to become within reach.

Abnormal social behavior is a symptom of many human conditions, including Fetal Alcohol Spectrum Disorders (FASD). The zebrafish may be an excellent model to study embryonic alcohol effects, owing to the ease of drug administration. In recent decades, zebrafish have become increasingly popular in behavioral neuroscience, and their elaborate behavioral repertoire can provide insights in brain functioning and social behavior in response to teratogens or pharmaceutical agents. Shoaling is the zebrafish’s most notable behavioral feature, but has not been well characterized. I have started this characterization by describing the ontogeny of shoaling behavior. Embryonic exposure to low doses of ethanol has been shown to impair social behavior without any gross morphological alterations in zebrafish. However, this has not been studied in freely moving groups (shoals). Validation for using a shoaling task in behavioral testing, and potentially for high throughput analyses in the future, hinges on thorough characterization of the behavioral effects, however subtle in alcohol treated zebrafish. In my studies, I have shown that social behavior is impaired in zebrafish in a group setting after one time embryonic ethanol exposure. I have also discovered that this impairment is accompanied by a reduction of dopamine, serotonin, DOPAC and 5HIAA, neurochemicals measured in whole brain samples using HPLC. I have developed refined methods of measuring shoaling behavior in ten member zebrafish groups, laying the foundation for high throughput screening of adult zebrafish. Zebrafish are an economical model, and therefore lend themselves particularly well for high throughput screening. However, current paradigms are still labor intensive and require substantial human capital. By refining current behavioral tests and deploying new analytical tools, high throughput screening is starting to become within reach.