Master of Science / Department of Psychology / Mary E. Cain / Rats reared in an enriched condition (EC) with novel stimuli and social contact with cohorts display less sensitization to nicotine than rats reared under impoverished conditions (IC). However, it is currently unknown what effect differential rearing has on nicotine-induced conditioned hyperactivity. The present study determined whether differential rearing affects conditioning to a nicotine-associated context. In addition, this study also examined the effects of mecamylamine, an antagonist to nicotinic acetylcholine receptors, on conditioned hyperactivity and sensitization. This antagonistic drug has been shown to attenuate the locomotor effects of nicotine. In the current study, EC, IC, and social condition (SC) rats were reared from 21 to 51 days of age before training for the acquisition of conditioned hyperactivity and sensitization. Nicotine (0.4 mg/kg) was administered prior to 1-h locomotor sessions. Conditioned hyperactivity testing followed. Rats then received 5 sessions of sensitization training followed by a 16-day drug-free rest period before being tested for sensitization. Mecamylamine (1.0 mg/kg) was administered to rats prior to the conditioned hyperactivity test and sensitization test. Nicotine treatment resulted in sensitization and conditioned hyperactivity in all differential rearing groups. EC rats displayed less locomotor activity in response to nicotine than both IC and SC rats. Pretreatment with mecamylamine blocked the expression of conditioned hyperactivity in EC and SC rats and attenuated sensitization in all three rearing groups. These findings suggest that environmental enrichment may alter nAChR binding during development and may be a protective factor in the initiation and relapse of smoking behavior.

The duodenal mucosa is regularly exposed to acid, digestive enzymes and ingested noxious agents. It is thus critical to maintain a protective barrier to prevent the development of mucosal injury and inflammation, which are often observed in situations when barrier function is impaired. The rate of mucosal bicarbonate secretion, the regulation of epithelial paracellular permeability and motility are each key components of duodenal barrier function. The hormone melatonin is present in high levels in the gastrointestinal tract and it has been hypothesized that melatonin exerts protective properties. This thesis aims to investigate the impact of exogenous melatonin on the regulation of duodenal barrier function and motility in anesthetized rats in vivo. In addition, duodenal tissue was examined histologically and the expression levels of tight junction proteins and melatonin receptors were assessed with qRT-PCR. It was found that melatonin stimulated mucosal bicarbonate secretion and decreased basal paracellular permeability. Exposing the duodenal mucosa to the well-characterized barrier breaker ethanol increased mucosal bicarbonate secretion, paracellular permeability and motility. Omission of luminal Clˉ abolished, while pretreatment with a nicotinic receptor antagonist reduced, the ethanol-induced bicarbonate secretion suggesting that the secretory response to ethanol is meditated via Clˉ/HCO3ˉexchangers and enteric neural pathways. Melatonin reduced the ethanol-induced increases in paracellular permeability and motility either when injected intravenously or when administered in drinking water for two weeks. The actions of melatonin were abolished by the melatonin receptor antagonist luzindole and by nicotinic acetylcholine receptor inhibition. Two weeks oral administration of melatonin up-regulated the expression levels of melatonin receptors, down-regulated the expression of ZO-3 while the expression of ZO-1, ZO-2, claudin 2-4, occludin and myosin light chain kinase were unaffected. Superficial epithelial changes in a few villi were seen in response to ethanol exposure, an effect that was histologically unchanged by melatonin pretreatment. In conclusion, the results suggest that melatonin plays an important role in the neurohumoral regulation of gastrointestinal mucosal barrier function and motility via receptor- and enteric neural-dependent pathways in vivo in rats. Melatonin might be a candidate for treatment of barrier dysfunction in humans.

Made available in DSpace on 2016-06-02T20:19:09Z (GMT). No. of bitstreams: 1 DissOSMB.pdf: 768691 bytes, checksum: a1ee980e3f11530bc253a291728b319e (MD5) Previous issue date: 2003-11-27 / Financiadora de Estudos e Projetos / Nicotine is a neuromodulator of the central nervous system, which has a wide variety of behavioural effects. The aim of this study is to investigate the role of nicotine and mecamylamine on the learning and memory mechanism through conditioning by a positive reinforcement process in 158 fish of the species Carassius auratus. For this purpose a rectangular aquarium was used (30×10×17 cm for total length, width and height, respectively). The aquarium was divided into two compartments, one black and one white. The experiment was done in five days: on the first day: habituation; on the second day: the natural preference of the animal was registered; on the third day: raining session number 1(T1), the animals were subjected to reinforcement in the white compartment; on the fourth day: training session number 2 (T2), the animals were subjected to reinforcement in the white compartment and were injected immediately after training; on the fifth day: test, the preference of the animals between the two compartments was registered once more. The animals were divided into eight groups according to the treatment. Four groups were injected immediately after training: vehicle (n=25); nicotine 2.0mg/kg (n=24); mecamylamine 1.0mg/kg (n=25); mecamylamine 2.0mg/kg (n=24); and four groups were injected three hours after training: vehicle (n=15); nicotine 2.0mg/kg; mecamylamine 1.0mg/kg (n=15); mecamylamine 2.0mg/kg (n=15). The statistical analysis was carried out using a two-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test or the Friedman test. The variables submitted to the analysis were: the time spent in each compartment; the latency and the number of times the fish crossed from one compartment to the other. The results show that in the groups, which were injected immediately the animals, showed a natural preference for the black compartment before training. In the post-training, the vehicle and mecamylamine 1,0mg/kg groups showed less preference for the black compartment, indicating learning. In the nicotine group there was a significant difference (P<0.01) between the time spent in the black and white compartments, however the animals of this group maintained the preference for the dark environment. In the mecamylamine 2.0mg/kg group, the animals changed the preference between the compartments showing facilitation of the learning process. In relation to latency the animals of the vehicle, mecamylamine 1.0 mg/kg and 2.0mg/kg groups, presented a significant reduction (P<0.05) of the L between the days showing that the animals learned by positive reinforcement and retained the task on the day of the test. In the animals of the nicotine group, there was a significant difference (P<0.05) of latency just between the T2 and the test days, showing a slower learning process. In relation to the number of times the fish crossed from one compartment to the other there was not a significant difference between the groups treated, suggesting that the drugs had no effect on the locomotor activity of the animals. In the groups injected after three hours, the animals still preferred the black compartment, showing that there was not any pro-active effect of the drugs on the memory consolidation process. With regards to latency, our results suggest that there was cholinergic drug action on unspecific neural systems. Thus, it suggests that concerning the immediate groups in this model there was an effect on the learning process. / A nicotina é um neuromodulador do sistema nervoso central que produz ampla variedade de efeitos comportamentais. A proposta deste estudo foi investigar o papel da nicotina e da mecamelamina sobre o mecanismo de aprendizagem e memória, através de condicionamento por reforço positivo, em peixes da espécie Carassius auratus. Foram utilizados 158 animais; um aquário retangular, com dimensões de 30 cm de comprimento, 10 cm de largura e 17 cm de altura, dividido ao meio em dois compartimentos, um preto e outro branco. O experimento foi realizado em cinco dias: dia I-habituação; dia II-registrouse a preferência natural do animal; dia III-treino 1, os animais receberam o reforço no compartimento branco; dia IV-treino 2, os animais receberam o reforço no compartimento branco e em seguida foram injetados; dia V-teste, foi observada a preferência do animal entre os dois compartimentos. Oito grupos de animais foram formados de acordo com a droga administrada. Quatro grupos injetados imediatamente no pós-treino: veículo (n=25); nicotina 2,0mg/kg (n=24); mecamelamina 1mg/kg (n=25); mecamelamina 2,0mg/kg (n=24); e quatro grupos injetados três horas após o treino: veículo (n=15); nicotina 2,0mg/kg (n=15); mecamelamina 1,0mg/kg (n=15); mecamelamina 2,0mg/kg (n=15). A análise estatística foi feita através do ANOVA de 2 vias seguida do teste Student-Newman- Keuls ou através do teste Friedman. As variáveis submetidas à análise foram o tempo de permanência (TP) em cada compartimento, a latência (L); e o número de cruzamentos (CZ). Os resultados mostram que nos grupos injetados imediatamente no pós-treino, os animais de todos os grupos tratados exibiram preferência natural pelo ambiente escuro. No pós-treino, os grupos veículo e mecamelamina 1mg/kg reduziram a preferência pelo compartimento preto, permitindo inferir aprendizagem. No grupo nicotina houve diferença significativa (p<0,01) entre a permanência nos compartimentos preto e branco, porém os animais mantiveram a preferência pelo ambiente escuro. No grupo mecamelamina 2mg/kg, os animais inverteram a preferência entre os compartimentos, indicando facilitação da aprendizagem em relação ao grupo veículo. Em relação à L, nos animais dos grupos veículo, mecamelamina 1,0mg/kg e 2,0mg/kg, a redução significativa (p<0,05) da L entre os dias indica que os animais aprenderam por reforço positivo e retiveram a tarefa no dia do teste. Nos animais do grupo nicotina, houve diferença significativa (p<0,05) da latência apenas entre os dias T2 e o Teste. Quanto ao número de CZ, não houve diferença significativa entre os grupos tratados, sugerindo que as drogas não exerceram efeito sobre a atividade locomotora dos animais. Nos grupos injetados após três horas (animais injetados três horas após o treino), a análise do TP no pós-treino indica que os animais continuaram preferindo o compartimento preto, indicando que não houve efeito pró-ativo das drogas sobre o processo de consolidação da memória. Quanto à L, nossos resultados sugerem que provavelmente, houve ações das drogas colinérgicas sobre sistemas neurais inespecíficos. Em relação ao número de CZ, não houve diferença significativa entre os grupos tratados, indicando que as drogas não exerceram efeito sobre a atividade locomotora do animal. Assim, sugere-se que nesse modelo, nos grupos imediatos, houve efeito da mecamelamina sobre a aprendizagem.

The focus of this study was to analyze whether the psychostimulant nicotine would enhance reference and working memory consolidation in rats tested on the 8-arm radial arm maze. Mecamylamine, a nicotine antagonist, was used to attempt to block the enhancement of memory consolidation. All rats were given one training trial/day for 12 consecutive days, and 4 arms were baited. Rats were separated into five groups: the saline-nicotine group received an intraperitoneal (i.p.) injection of saline immediately after each trial followed 15 min later by an subcutaneous (s.c.) injection of nicotine (0.6 mg/kg free base); the nicotine-delay group received an s.c. injection of nicotine 2 h after each training trial, two groups received an i. p. injection of one of two different doses of mecamylamine (2.5 and 6.0 mg/kg) immediately after each trial, which was followed 15 min later by an s.c. nicotine injection, and a control group received an i.p. injection of saline immediately and 15 min after each training trial. Results showed that the saline-nicotine group made fewer reference and working memory errors than the saline- or nicotine-delay groups, but only the effect of nicotine on reference memory was blocked by the higher dose of mecamylamine. It appears from these results that nicotine's effects on reference and working memory may be mediated through different mechanisms.

Prepulse inhibition (PPI) is an operational measure of sensorimotor gating and is known to be reduced when the dopamine D2 receptor is activated. We used a rodent model of psychosis in which increases in dopamine D2 receptor sensitivity are produced through neonatal quinpirole (a dopamine D2 / D3 agonist) treatment to rats. Rats were administered quinpirole (1mg/kg) or saline from postnatal day (P) 1-21. Rats were raised to adulthood and tested on PPI. Results showed that neonatal quinpirole treatment produced a significant reduction in PPI, and nicotine exacerbated this reduction. This reduction was partially blocked by the nicotinic antagonist mecamylamine. Brain tissue was analyzed for regulators of G-protein signaling (RGS) and results showed that neonatal quinpirole significantly decreased RGS9, but increased RGS17 as compared to controls. These results appear to indicate that the G-protein couples more efficiently to the D2 receptor, and nicotine exacerbates PPI deficits in D2 receptor-primed rats.

Consumption of alcohol and tobacco is common all around the world and these drugs are frequently consumed concomitantly. It has been estimated that 70-80 % of alcoholics are smokers and non-alcoholic drinkers are more often smokers than teetotallers. Alcohol and tobacco may affect the risk of developing neurological diseases and might influence this risk differently when combined compared to when only one of these compounds is consumed. Some in vitro-research have shown that non-toxic concentrations of ethanol and nicotine, in combination, can exert toxicity, and might do so in a synergistic way. In this work, investigations were made to see if the neuronal nicotinic acetylcholine receptors (nAChRs) and NMDA receptors are involved in this interactive behaviour between ethanol and nicotine. A human neuroblastoma SH-SY5Y cell line was treated with ethanol and nicotine at different concentrations and cell viability was measured through an MTT-assay. A significant reduction in cell viability was induced by chronic treatment with a low-dose combination of ethanol and nicotine. The cell viability reduction was completely inhibited by pretreatment with the non-specific nAChR antagonist mecamylamine. This suggests that nAChRs are involved in low-dose ethanol-nicotine interactions. The NMDA receptor antagonist memantine did not affect the ethanol-nicotine effect, which implies that NMDA receptors are not involved in low-dose ethanol-nicotine interactions in SH-SY5Y cells. However, it is unclear if the SH-SY5Y cell line expresses fully functional NMDA receptors. The expression of NMDA receptors might vary with cell passage number. Further research has to be done to uncover the contribution of specific nAChR subtypes to the ethanol-nicotine interaction. There also remains to be revealed if human neuroblastoma SH-SY5Y cells express fully functional NMDA receptors and how cell passage number affects the expression of these receptors.

Rationale: Peak initiation of smoking occurs during adolescence and early onset of smoking is associated with a reduced probability of quitting and greater risk of relapse compared to later onset. Considering the epidemiological evidence, adolescents may exhibit a unique biological susceptibility to nicotine taking, in addition to the behavioural and psychosocial factors known to influence adolescent smoking. Objectives: The current series of experiments, using a rat model of adolescent nicotine taking, was designed to investigate age differences in the processes involved in the acquisition and maintenance of nicotine taking that might account for the elevated initiation rates of smoking during adolescence. Methods: We first investigated age differences in the neural response to acute nicotine administration using c-fos mRNA expression. We then examined age differences in the rewarding and aversive effects of nicotine in the conditioned place preference (CPP) and conditioned taste avoidance (CTA) paradigms, respectively. The direct reinforcing effects of nicotine were tested in adolescent and adult rats under a variety of reinforcement schedules in the operant intravenous self-administration paradigm; extinction and nicotine priming-induced reinstatement were also examined. Finally, age differences in nicotine withdrawal precipitated by mecamylamine were assessed. Results: Nicotine had greater activational effects on c-fos mRNA expression in reward-related neural substrates of adolescent compared to adult brain. Adolescent rats were also more sensitive to the rewarding effects of nicotine (CPP) yet less sensitive to its aversive effects (CTA) compared to adult rats. Nicotine was equally reinforcing in adolescents and adults self-administering under simple reinforcement schedules, but adults were more motivated to obtain nicotine under higher reinforcement schedules. Adults were more resistant to extinction, yet both age groups demonstrated similar priming-induced reinstatement of nicotine seeking. Under spontaneous acquisition conditions, adults were more sensitive to the reinforcing effects of a low nicotine infusion dose. The aversive effects of nicotine withdrawal were also more prominent in adults compared to adolescents. Conclusions: These findings have important implications since they demonstrate a unique susceptibility to the conditioned rewarding effects of nicotine that would promote acquisition of smoking behaviour during adolescence, whereas adults may be more vulnerable to processes involved in its maintenance.

Rationale: Peak initiation of smoking occurs during adolescence and early onset of smoking is associated with a reduced probability of quitting and greater risk of relapse compared to later onset. Considering the epidemiological evidence, adolescents may exhibit a unique biological susceptibility to nicotine taking, in addition to the behavioural and psychosocial factors known to influence adolescent smoking. Objectives: The current series of experiments, using a rat model of adolescent nicotine taking, was designed to investigate age differences in the processes involved in the acquisition and maintenance of nicotine taking that might account for the elevated initiation rates of smoking during adolescence. Methods: We first investigated age differences in the neural response to acute nicotine administration using c-fos mRNA expression. We then examined age differences in the rewarding and aversive effects of nicotine in the conditioned place preference (CPP) and conditioned taste avoidance (CTA) paradigms, respectively. The direct reinforcing effects of nicotine were tested in adolescent and adult rats under a variety of reinforcement schedules in the operant intravenous self-administration paradigm; extinction and nicotine priming-induced reinstatement were also examined. Finally, age differences in nicotine withdrawal precipitated by mecamylamine were assessed. Results: Nicotine had greater activational effects on c-fos mRNA expression in reward-related neural substrates of adolescent compared to adult brain. Adolescent rats were also more sensitive to the rewarding effects of nicotine (CPP) yet less sensitive to its aversive effects (CTA) compared to adult rats. Nicotine was equally reinforcing in adolescents and adults self-administering under simple reinforcement schedules, but adults were more motivated to obtain nicotine under higher reinforcement schedules. Adults were more resistant to extinction, yet both age groups demonstrated similar priming-induced reinstatement of nicotine seeking. Under spontaneous acquisition conditions, adults were more sensitive to the reinforcing effects of a low nicotine infusion dose. The aversive effects of nicotine withdrawal were also more prominent in adults compared to adolescents. Conclusions: These findings have important implications since they demonstrate a unique susceptibility to the conditioned rewarding effects of nicotine that would promote acquisition of smoking behaviour during adolescence, whereas adults may be more vulnerable to processes involved in its maintenance.

Abdominal surgery inhibits gastrointestinal motility, a phenomenon referred to as postoperative ileus. Since the postoperative ileus disturbs duodenal physiology it is important to minimize the side effects of this condition. Recent experiments in our laboratory show that treatment of anaesthetised rats with parecoxib, a selective cyclooxygenase-2 inhibitor, prevents duodenal postoperative ileus, increases duodenal mucosal bicarbonate secretion and improves other functions as well. One aim of the thesis was to investigate whether removal of luminal chloride affect the parecoxib- and the vasoactive intestinal peptide (VIP)-induced stimulation of duodenal mucosal bicarbonate secretion. The proximal duodenum of anaesthetised Dark Agouti rats was perfused with isotonic solutions containing zero or low Cl- and the effect on luminal alkalinisation determined. The basal as well as the parecoxib-induced increase in alkalinisation, but not that stimulated by VIP, were markedly reduced in the absence of luminal Cl-. One important function of the duodenum is to adjust luminal osmolality towards that in the blood. It is believed that the adjustment of osmolality in the duodenum is achieved by osmosis and diffusion of electrolytes along their concentration gradients and that these processes occur predominately paracellularly. Another aim of the thesis was to examine whether prevention of postoperative ileus affects the duodenal response to luminal hypertonicity. The proximal duodenum of anaesthetised Dark Agouti and Sprague-Dawley rats were perfused with hypertonic solutions of different composition and osmolality and the effects on duodenal motility, alkaline secretion, transepithelial fluid flux, mucosal permeability and the adjustment of luminal osmolality were determined in absence and presence of parecoxib. It is concluded that COX-2 inhibition increases duodenal mucosal bicarbonate secretion by stimulating apical Cl-/HCO3- exchange in duodenocytes. Furthermore, pretreatment of anaesthetised rats with parecoxib improves a number of duodenal functions in both rat strains that contribute to improve the ability to adjust luminal osmolality. The choice of rat strain is another important feature to consider when interpreting the results because the DA strain was more responsive to luminal hypertonicity than the SD strain. Finally, several evidences are provided to suggest that the adjustment of luminal osmolality in the rat duodenum is a regulated process.