Excessive and compulsive ethanol drinking is one of the most serious public health issues. Therefore, it is vital to increase the knowledge about risks and protection for alcohol use disorders (AUD) to optimize prevention and treatment strategies. Ethanol consumption commonly initiates during adolescence when extensive neuronal maturation and development also occurs. Early exposure to ethanol is a risk factor for AUD, but the effects of adolescent drinking and the basis for the individual susceptibility to AUD are not fully understood. The interactions between genotype and environmental factors determine the individual risk for AUD and this thesis aimed to examine the environmental impact. The specific aims were to investigate 1) how early-life conditions affect adolescent voluntary ethanol drinking, behavioural profiles, endogenous opioids and response to treatment with an opioid antagonist (naltrexone), and 2) whether alterations detected in the offspring may be mediated by variations in maternal behaviour. A rodent maternal separation (MS) model was used to mimic a protective and risk-inducing early-life environment, respectively, with the use of 15 min (MS15) or 360 min (MS360) of daily MS. The main findings were 1) the MS360, but not the MS15 rats, responded to naltrexone following adolescent ethanol drinking; all adolescent rats had a high voluntary ethanol intake independent of early environmental conditions whereas in the adult groups the MS360, but not the MS15 rats, increased their ethanol intake and preference over time; adolescent ethanol exposure resulted in higher dynorphin levels in hippocampus and higher Met-enkephalin-Arg6Phe7 in the amygdala, independently of rearing conditions, 2) behavioural profiling using the multivariate concentric square field™ test showed: the young MS360 rats had increased risk assessment and risk taking behaviour compared to the young MS15 rats; the young MS15 rats increased, whereas the young MS360 rats decreased, their risk assessment and risk taking behaviour over time; differences in pup-retrieval strategies where the MS360 dams retrieved some pups into a safe area but as compared to MS15 rats they left more pups in a risk area; increased risk assessment behaviour in the MS360 dams immediately after weaning. Taken together, early-life environmental conditions alter adult but not adolescent drinking, the response to naltrexone, and behaviour in dams and offspring. Adolescent rats consumed more ethanol independent of rearing conditions and displayed increased opioid levels in brain areas related to cognition and addiction.

Ο νεογνικός χειρισμός, ένα πειραματικό μοντέλο πρώιμων εμπειριών, είναι γνωστό οτι επηρεάζει τη λειτουργία του άξονα υποθαλάμου-υπόφυσης-επινεφριδίων, βελτιώνοντας έτσι την προσαρμοστικότητα, την αντιμετώπιση του στρες, τις διανοητικές ικανότητες και γενικά τις διεργασίες του εγκεφάλου που σχετίζονται με την πλαστικότητα. Προηγούμενη μελέτη έχει δείξει οτι ο νεογνικός χειρισμός παρουσιάζει εκλεκτικές επιδράσεις στους υποδοχείς του N-μεθυλο-D-ασπαρτικού οξέος (NMDA) του διεγερτικού νευροδιαβιβαστή γλουταμινικό στον εγκέφαλο των επίμυων. Οι υποδοχείς του γλουταμινικού που διαμεσολαβούν την ταχεία συναπτική νευροδιαβίβαση στις διεγερτικές συνάψεις του κεντρικού νευρικού συστήματος είναι οι υποδοχείς του α-αμινο-3-υδροξυ-5-μεθυλο-ισοξαζολο-προπιονικού οξέος (AMPA) και είναι κρίσιμοι κατά τη νευρωνική ανάπτυξη, τη συναπτική πλαστικότητα και τη δομική αναδιαμόρφωση του εγκεφάλου. Συντίθενται από τέσσερις υπομονάδες, τις GluRA, GluRB, GluRC και GluRD, οι οποίες συνδυάζονται για να σχηματίσουν τετραμερή. Οι περισσότεροι υποδοχείς AMPA είναι ετεροτετραμερή, αποτελούμενα από τουλάχιστον δύο από τις αναφερθείσες υπομονάδες. Οι υποδοχείς AMPA που είναι διαβατοί στο ασβέστιο δεν περιέχουν την υπομονάδα GluRB, ενώ τόσο η υπομονάδα GluRA, όσο και η GluRB, παίζουν σημαντικό ρόλο στη συναπτική διακίνηση των υποδοχέων AMPA. Ο σκοπός της παρούσας εργασίας ήταν να ερευνηθεί κατά πόσο ο νεογνικός χειρισμός μπορεί να επιδρά στους υποδοχείς AMPA, εφόσον έχει δειχθεί οτι η σύνθεση των υπομονάδων και κατά συνέπεια και οι ιδιότητες των υποδοχέων AMPA στη σύναψη μεταβάλλονται από την αισθητική εμπειρία και κατά πόσο αυτή η επίδραση είναι εξαρτώμενη από το φύλο. Σύμφωνα με το παρόν πρωτόκολλο νεογνικού χειρισμού, κάθε νεογνό απομακρυνόταν από τη φωλιά για 15 λεπτά καθημερινά από την πρώτη μεταγεννητική ημέρα μέχρι τον απογαλακτισμό του τρεις εβδομάδες μετά. Η τεχνική του in situ υβριδισμού χρησιμοποιήθηκε για την εντόπιση και ποσοτικοποίηση της έκφρασης του mRNA της κάθε υπομονάδας. Η έκφραση των υπομονάδων του υποδοχέα AMPA μελετήθηκε σε συγκεκριμένες εγκεφαλικές περιοχές που εμπλέκονται στο συναίσθημα, στη μάθηση, στη μνήμη και στην αισθητική αντίληψη, όπως ο ιππόκαμπος, η αμυγδαλή και ο εγκεφαλικός φλοιός ενήλικων αρσενικών και θηλυκών επίμυων. Τα αποτελέσματα της έρευνας έδειξαν οτι ο νεογνικός χειρισμός προκάλεσε διαφορετικές και σε κάποιες περιπτώσεις φυλετικά διμορφικές μεταβολές στην έκφραση των υπομονάδων του υποδοχέα AMPA, με τρόπο ειδικό ανά υπομονάδα και περιοχή. Πιο συγκεκριμένα, τα ζώα που είχαν υποστεί νεογνικό χειρισμό, αρσενικά και θηλυκά, είχαν μειωμένα επίπεδα mRNA της GluRB και της GluRC στο ραχιαίο ιππόκαμπο και στο σωματαισθητικό φλοιό, συγκριτικά με τα ζώα χωρίς χειρισμό. Επιπλέον, τα επίπεδα mRNA της GluRC ήταν μειωμένα στην αμυγδαλή και στον προμετωπιαίο φλοιό και τα επίπεδα mRNA της GluRD ήταν μειωμένα στον κοιλιακό ιππόκαμπο αρσενικών και θηλυκών ζώων που είχαν υποστεί χειρισμό. Επιπροσθέτως, ο νεογνικός χειρισμός είχε φυλετικά διμορφικές επιδράσεις, αυξάνοντας τα επίπεδα mRNA της GluRA στο ραχιαίο ιππόκαμπο των αρσενικών ζώων και μειώνοντάς τα στα θηλυκά, καθώς και μειώνοντας τα επίπεδα mRNA της GluRΒ στον κοιλιακό ιππόκαμπο και στην αμυγδαλή μόνο των θηλυκών επίμυων. Οι μεταβολές στο mRNA των υποδοχέων AMPA θεωρούμε οτι συντελούνται μέσω επιγενετικής ρύθμισης λόγω του νεογνικού χειρισμού. Τα αποτελέσματα μας στο ραχιαίο ιππόκαμπο υποδεικνύουν ότι ο νεογνικός χειρισμός είναι πιθανό να προκαλεί επαγόμενη από την εμπειρία συναπτική ενδυνάμωση και αποδυνάμωση αρσενικών και θηλυκών ζώων αντίστοιχα, δεδομένων των αντίθετων μεταβολών που παρατηρούνται στα επίπεδα του mRNA της υπομονάδας GluRA στα δύο φύλα. Επιπλέον, η παρατηρούμενη μείωση της έκφρασης του mRNA της GluRB πιθανόν να οδηγεί σε μειωμένο αριθμό αδιάβατων στο ασβέστιο υποδοχέων AMPA που περιέχουν την υπομονάδα GluRB, οπότε ο νεογνικός χειρισμός μπορεί να προκαλεί μεταβολή στο φαινότυπο του υποδοχέα AMPA από αδιάβατο σε διαβατό στο ασβέστιο και τελικά αύξηση της πλαστικότητας της σύναψης στις περιοχές όπου παρατηρούνται οι μεταβολές. Συμπερασματικά, στην παρούσα μελέτη δείξαμε οτι οι υπομονάδες του υποδοχέα AMPA μεταβάλλονται από το νεογνικό χειρισμό, ένα μοντέλο πρώιμης εμπειρίας, στον εγκέφαλο ενήλικων επίμυων, με τρόπο ειδικό ανά υπομονάδα και ανά περιοχή και σε κάποιες περιπτώσεις φυλετικά διμορφικό. Αυτό σημαίνει οτι η γλουταμινεργική νευροδιαβίβαση μεταβάλλεται από μια πρώιμη εμπειρία. Αυτή η επίδραση του νεογνικού χειρισμού μπορεί να είναι ένας από τους παράγοντες που υπόκεινται της αυξημένης πλαστικότητας του εγκεφάλου των ζώων που έχουν υποστεί νεογνικό χειρισμό, η οποία εκδηλώνεται τόσο σε κυτταρικό επίπεδο όσο και στο συμπεριφορικό επίπεδο. / Neonatal handling, an experimental model of early life experiences, is known to affect the hypo¬thalamic-pituitary-adrenal axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related pro¬cesses. Previous study has shown selective effects of neonatal handling on rat brain NMDA receptors. AMPA receptors (AMPARs) mediate fast synaptic trans¬mission at excitatory synapses in the CNS and are crucial during neuro¬nal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as GluRA, GluRB, GluRC and GluRD, which combine to form tetramers. Most AMPARs are heterotetramerics, made of at least two of the four pro¬per subunits GluRA-D. AMPA receptors that are permeable to Ca2+ lack the GluRB subunit, while both GluRA and GluRB subunits have an important role in AMPAR trafficking towards the synapse. The present study addressed the question of whether neonatal handling might have an effect on AMRARs, since it has been shown that the subunit composition and thus the synaptic properties of AMPARs, changes in response to sensory experience, and whether this effect is gender-specific. According to the current neonatal handling pro¬tocol, each pup of a litter was removed from the nest for 15 min daily from the first postnatal day 1 (PND1) until weaning (PND22). In situ hybridization was used in order to localize and quantify subunit mRNA expression, with specific cDNA oligo¬nucleotides. AMPAR subunit expression was studied in specific brain regions that are involved in emotions, learning, memory and sensory perception, such as the hippo¬campus, cerebral cortex and amydgala of adult male and female rats. We found that neonatal handling caused differential and in cases sexually dimorphic changes in AMPA receptor subunit expression, depending on the brain region and the subunit. More specifically, neonatally handled animals, both males and females, had lower GluRB and GluRC mRNA levels in the dorsal hippocampus and the somatosensory cortex, compared to the non-handled. Moreover, GluRC mRNA levels were decreased in the amygdala and the prefrontal cortex and GluRD mRNA levels were decreased in the ventral hippocampus of handled animals of both sexes. Furthermore, neonatal handling had sexually dimorphic effects, increasing GluRA mRNA levels in the dorsal hippocampus of males while decreasing them in females, as well as decreasing GluRB mRNA levels in the ventral hippocampus and the amygdala only of the females. The observed changes in AMPA receptor mRNA levels are thought to occur via epigenetic regulation in response to neonatal handling. Our results in dorsal hippocampus suggest that neonatal handling may induce experience-dependent synaptic strengthening in males and weakening in females, due to the opposite changes observed in GluRA mRNA levels of the two sexes. Moreover, the observed decrease in GluRB mRNA expression most likely leads to reduced GluRB-containing calcium-impermeable AMPA receptors, therefore neonatal handling may cause a switch in AMPA receptor phenotype from calcium-impermeable to calcium-permeable AMPA receptors, and thus induce a higher synaptic plasticity in the regions where the changes are observed. In the present study we show that neonatal handling, an experimental model of early life experiences, induces changes in the AMPA receptor subunits expression in the adult rat brain that are region- and subunit-specific and in cases sexually dimorphic. These results demonstrate that glutamatergic transmission changes in response to an early experience. This effect of handling could be one of the factors underlying the increased plasticity of the brain of neonatally handled animals, which is manifested both at the cellular and at the behavioural/systemic level.

Estudos prévios demonstraram que ratos manipulados no período neonatal apresentavam um maior consumo de alimento doce na vida adulta, em comparação com ratos não manipulados, quando esse alimento lhes é oferecido durante curtos períodos de tempo. O objetivo deste trabalho foi avaliar os efeitos da manipulação neonatal sobre o padrão metabólico de animais adultos expostos cronicamente a uma dieta hipercalórica e palatável (chocolate). Desta forma, foram medidos o consumo de alimentos (ração padrão e chocolate), ganho de peso corporal, depósito de gordura abdominal, peso das glândulas adrenais e parâmetros bioquímicos como lipídios, glicose, insulina, e corticosterona plasmáticos em ratos adultos que foram expostos ou não à manipulação neonatal (10 min/dia, nos 10 primeiros dias de vida). Ratos que foram manipulados no período neonatal apresentaram uma ingestão de chocolate aumentada, porém este consumo diminuiu com o passar do tempo, atingindo níveis semelhantes aos dos animais não manipulados. Ratos machos manipulados apresentaram maior peso corporal, assim como maior eficiência calórica e níveis de triglicerídeos diminuídos no plasma, independentemente da dieta, enquanto que ratas fêmeas não manipuladas que receberam chocolate apresentaram depósito de gordura abdominal aumentado. Também foi observado, nas ratas fêmeas, maior depósito de gordura abdominal, níveis mais elevados de colesterol total e de glicose, bem como níveis mais baixos de insulina, quando comparadas com ratos machos. O consumo de chocolate reduziu o peso das glândulas adrenais tanto nos animais manipulados quanto nos controles. Assim, esses achados sugerem que a manipulação neonatal induz um padrão metabólico particular, o qual parece proteger os animais dos efeitos deletérios de uma dieta hiperpalatável e hipercalórica, e ainda, a manipulação parece afetar diferentemente ratos machos e fêmeas. / Previous studies have already observed that neonatal handled rats consume more sweet food compared to non-handled ones in adult life, when this type of food is offered during short intervals of time. The aim, in this study, was to assess the effects of neonatal handling on the metabolic pattern of adult rats chronically exposed to a palatable diet (chocolate). We measured the consumption of foods (standard lab chow and chocolate), body weight gain, abdominal fat deposition, adrenal glands’ weight and biochemical measures such as plasma lipids, glucose, insulin, and corticosterone in adult rats which had been exposed or not to neonatal handling (10 min/day, 10 first days of life). An increased intake of chocolate in handled rats was found, but this consumption decreases as the days passed by, reaching the same levels as non-handled animals. Handled male animals presented higher body weight, higher caloric efficiency and lower plasma triglycerides levels, independently of the diet, while non-handled females rats receiving chocolate presented increased abdominal fat deposition. Female rats also presented increased abdominal fat deposition, higher total cholesterol and glucose levels as well as lower insulin in comparison to males. Interestingly, chocolate consumption diminished adrenal glands’ weight in both groups. These findings suggest that neonatal handling induces a particular metabolic pattern, which seems to protect the animals against deleterious effects of a highly palatable, highly caloric diet, and also it seems to differentially affect females and males.

Early life events, in both humans and animals, may influence on brain development, leading to long-term consequences on adult behavior. Thus, this study was carried out in order to assess the influence of neonatal tactile stimulation (TS), when associated to sub-therapeutic administration of sertraline (SERT) in rats, on behavioral parameters related to depression-like. We also evaluated the influence of the TS-SERT association on the hypothalamic-pituitary-adrenal axis, as well as on the development of anxiety-like symptoms, assessed in different behavioral. Male rat pups were daily submitted to 10 min TS, from postnatal day (PND) 8 to PND 14. On DPN50, rats were first exposed to forced swimming test (FST). Then, after 24h, it was conducted the SERT administration and, 30 minutes later, animals were submitted to new evaluation on FST. On DPN51, rats were evaluated on the elevated plus maze (EPM). The animals were euthanized 24h after the behavioral tasks, followed by biochemical assessments. TS per se decreased depression behaviors, since there was an increase on swimming time and a decrease on immobility time in FST. On the EPM task, TS diminished behaviors related to anxiety, as shown in the increase of time on open arms and decrease on anxiety rates. In addition, TS also decreased corticosterone and cortisol plasma levels. All these results were enhanced when TS was associated to a sub-therapeutic dose of SERT. From these results, we suggest that neonatal handling TS may accomplish beneficial influence on parameters of depression, promoting the ability to cope with stressful situations in adulthood. / Eventos ocorridos em estágios iniciais do desenvolvimento, tanto em humanos quanto em animais, influenciam o desenvolvimento do cérebro e podem ter consequências em longo prazo na idade adulta. Assim, o presente estudo foi realizado para avaliar a influência da estimulação tátil neonatal (ET), associada à administração de uma dose subterapêutica de sertralina (SERT), sobre parâmetros comportamentais relacionados a depressão em ratos. A influência da associação ET-SERT sobre o eixo hipotálamo-pituitária-adrenal, assim como sobre o desenvolvimento de comportamentos de ansiedade foram também avaliados. Filhotes machos de ratos Wistar foram diariamente submetidos à ET durante 10 min, desde o dia pós-natal (DPN) 8 até o DPN14. No DPN50, os animais foram previamente expostos ao nado forçado. Após 24h, receberam a administração de SERT e, decorridos 30 minutos, foram novamente avaliados mediante o estresse do nado forçado. No DPN 51, os animais também foram avaliados no labirinto em cruz elevado (LCE). Os animais foram eutanasiados 24h após os testes comportamentais, seguindo-se as avaliações bioquímicas. A ET per se reduziu comportamentos de depressão, tendo em vista a observação do maior tempo de nado e menor tempo de imobilidade no teste do nado forçado. No teste do LCE, a ET diminuiu comportamentos relacionados à ansiedade, como observado pelo maior tempo nos braços abertos e pelo menor índice de ansiedade. Além disso, animais expostos à ET apresentaram menores níveis plasmáticos de corticosterona e cortisol. Todos estes resultados foram potencializados quando a ET foi associada a uma dose subterapêutica de SERT. Considerando estes dados, é possível propor que a ET neonatal é capaz de exercer influências benéficas sobre parâmetros de depressão, favorecendo a capacidade para lidar com situações estressantes na vida adulta.

Estudos prévios demonstraram que ratos manipulados no período neonatal apresentavam um maior consumo de alimento doce na vida adulta, em comparação com ratos não manipulados, quando esse alimento lhes é oferecido durante curtos períodos de tempo. O objetivo deste trabalho foi avaliar os efeitos da manipulação neonatal sobre o padrão metabólico de animais adultos expostos cronicamente a uma dieta hipercalórica e palatável (chocolate). Desta forma, foram medidos o consumo de alimentos (ração padrão e chocolate), ganho de peso corporal, depósito de gordura abdominal, peso das glândulas adrenais e parâmetros bioquímicos como lipídios, glicose, insulina, e corticosterona plasmáticos em ratos adultos que foram expostos ou não à manipulação neonatal (10 min/dia, nos 10 primeiros dias de vida). Ratos que foram manipulados no período neonatal apresentaram uma ingestão de chocolate aumentada, porém este consumo diminuiu com o passar do tempo, atingindo níveis semelhantes aos dos animais não manipulados. Ratos machos manipulados apresentaram maior peso corporal, assim como maior eficiência calórica e níveis de triglicerídeos diminuídos no plasma, independentemente da dieta, enquanto que ratas fêmeas não manipuladas que receberam chocolate apresentaram depósito de gordura abdominal aumentado. Também foi observado, nas ratas fêmeas, maior depósito de gordura abdominal, níveis mais elevados de colesterol total e de glicose, bem como níveis mais baixos de insulina, quando comparadas com ratos machos. O consumo de chocolate reduziu o peso das glândulas adrenais tanto nos animais manipulados quanto nos controles. Assim, esses achados sugerem que a manipulação neonatal induz um padrão metabólico particular, o qual parece proteger os animais dos efeitos deletérios de uma dieta hiperpalatável e hipercalórica, e ainda, a manipulação parece afetar diferentemente ratos machos e fêmeas. / Previous studies have already observed that neonatal handled rats consume more sweet food compared to non-handled ones in adult life, when this type of food is offered during short intervals of time. The aim, in this study, was to assess the effects of neonatal handling on the metabolic pattern of adult rats chronically exposed to a palatable diet (chocolate). We measured the consumption of foods (standard lab chow and chocolate), body weight gain, abdominal fat deposition, adrenal glands’ weight and biochemical measures such as plasma lipids, glucose, insulin, and corticosterone in adult rats which had been exposed or not to neonatal handling (10 min/day, 10 first days of life). An increased intake of chocolate in handled rats was found, but this consumption decreases as the days passed by, reaching the same levels as non-handled animals. Handled male animals presented higher body weight, higher caloric efficiency and lower plasma triglycerides levels, independently of the diet, while non-handled females rats receiving chocolate presented increased abdominal fat deposition. Female rats also presented increased abdominal fat deposition, higher total cholesterol and glucose levels as well as lower insulin in comparison to males. Interestingly, chocolate consumption diminished adrenal glands’ weight in both groups. These findings suggest that neonatal handling induces a particular metabolic pattern, which seems to protect the animals against deleterious effects of a highly palatable, highly caloric diet, and also it seems to differentially affect females and males.

Estudos prévios demonstraram que ratos manipulados no período neonatal apresentavam um maior consumo de alimento doce na vida adulta, em comparação com ratos não manipulados, quando esse alimento lhes é oferecido durante curtos períodos de tempo. O objetivo deste trabalho foi avaliar os efeitos da manipulação neonatal sobre o padrão metabólico de animais adultos expostos cronicamente a uma dieta hipercalórica e palatável (chocolate). Desta forma, foram medidos o consumo de alimentos (ração padrão e chocolate), ganho de peso corporal, depósito de gordura abdominal, peso das glândulas adrenais e parâmetros bioquímicos como lipídios, glicose, insulina, e corticosterona plasmáticos em ratos adultos que foram expostos ou não à manipulação neonatal (10 min/dia, nos 10 primeiros dias de vida). Ratos que foram manipulados no período neonatal apresentaram uma ingestão de chocolate aumentada, porém este consumo diminuiu com o passar do tempo, atingindo níveis semelhantes aos dos animais não manipulados. Ratos machos manipulados apresentaram maior peso corporal, assim como maior eficiência calórica e níveis de triglicerídeos diminuídos no plasma, independentemente da dieta, enquanto que ratas fêmeas não manipuladas que receberam chocolate apresentaram depósito de gordura abdominal aumentado. Também foi observado, nas ratas fêmeas, maior depósito de gordura abdominal, níveis mais elevados de colesterol total e de glicose, bem como níveis mais baixos de insulina, quando comparadas com ratos machos. O consumo de chocolate reduziu o peso das glândulas adrenais tanto nos animais manipulados quanto nos controles. Assim, esses achados sugerem que a manipulação neonatal induz um padrão metabólico particular, o qual parece proteger os animais dos efeitos deletérios de uma dieta hiperpalatável e hipercalórica, e ainda, a manipulação parece afetar diferentemente ratos machos e fêmeas. / Previous studies have already observed that neonatal handled rats consume more sweet food compared to non-handled ones in adult life, when this type of food is offered during short intervals of time. The aim, in this study, was to assess the effects of neonatal handling on the metabolic pattern of adult rats chronically exposed to a palatable diet (chocolate). We measured the consumption of foods (standard lab chow and chocolate), body weight gain, abdominal fat deposition, adrenal glands’ weight and biochemical measures such as plasma lipids, glucose, insulin, and corticosterone in adult rats which had been exposed or not to neonatal handling (10 min/day, 10 first days of life). An increased intake of chocolate in handled rats was found, but this consumption decreases as the days passed by, reaching the same levels as non-handled animals. Handled male animals presented higher body weight, higher caloric efficiency and lower plasma triglycerides levels, independently of the diet, while non-handled females rats receiving chocolate presented increased abdominal fat deposition. Female rats also presented increased abdominal fat deposition, higher total cholesterol and glucose levels as well as lower insulin in comparison to males. Interestingly, chocolate consumption diminished adrenal glands’ weight in both groups. These findings suggest that neonatal handling induces a particular metabolic pattern, which seems to protect the animals against deleterious effects of a highly palatable, highly caloric diet, and also it seems to differentially affect females and males.

Made available in DSpace on 2016-04-29T13:17:48Z (GMT). No. of bitstreams: 1 Larissa Gomes Lacerda.pdf: 718140 bytes, checksum: b435ea39accd2e28173856939c3a4690 (MD5) Previous issue date: 2013-05-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Chronic Mild Stress (CMS) an experimental animal model of anhedonia induced by exposing the rats to a protocol of chronic stress for a long period of time. At the Laboratório do Programa de Pós Graduação em Psicologia Experimental: Análise do Comportamento, a research line about CMS and its relation with the operant behavior has been developed since 2001, as in Pereira (2009). Unlike other studies conducted in this laboratory, Pereira (2009) did not observe sucrose intake and preference reducing (anhedonia) along stressors. One pointed hypothesis was the neonatal handling was a responsible variable for that. The goal of this study was to investigate whether the neonatal handling alters the fluid intake and preference on rats that were submitted to the stressors protocol (CMS) during the adult phase. Three groups were divided among experimental conditions: 1) neonatal handling, 2) protocol and 3) neonatal handling and protocol. The used measures were: weight, ration and water intake and weekly tests over intake and fluid preference. Results found show that stressors protocol reduced weight of subjects from the manipulation group (MP) as for the ones from only stressors protocol group (P). Food and water intake also presented similar results for Groups P and MP: all subjects presented water intake enhance and ration intake reducing during protocol. For fluid intake and preference tests, both Group P and MP presented preference oscillation at no stressors period and sucrose intake enhance during protocol submission weeks, that means, they did not produced anhedonia. Results allow to assure that the group submitted to maternal manipulation with 15min mother s separation did not show any difference in comparison to the nohandling group, specially related to preference and intake tests, when submitted to CMS / Chronic Mild Stress (CMS) um modelo animal experimental de anedonia induzida através da exposição de ratos a um protocolo de estressores crônicos por um longo período de tempo. No Laboratório do Programa de Pós Graduação em Psicologia Experimental: Análise do Comportamento, uma linha de pesquisa sobre o CMS e a relação com o comportamento operante tem sido desenvolvida desde 2001, como em Pereira, 2009. Diferente de outros estudos realizados nesse laboratório, Pereira (2009) não observou redução da ingestão e preferência de sacarose (anedonia) durante os estressores. Uma das hipóteses levantadas foi que a manipulação neonatal tenha sido uma variável responsável por isso. O objetivo do presente estudo foi investigar se a manipulação neonatal, altera a ingestão e preferência de líquidos em ratos que passaram pelo protocolo de estressores (CMS) durante a fase adulta. Três grupos foram divididos entre as condições experimentais: 1) manipulação neonatal, 2) protocolo e 3) manipulação neonatal e protocolo. As medidas utilizadas foram: peso, consumo de ração e de água e testes semanais de ingestão e preferência de líquido. Os resultados encontrados mostram que o protocolo de estressores diminuiu o peso dos sujeitos tanto para o grupo que passou pela manipulação (MP) quanto para o que passou apenas pelo protocolo de estressores (P). O consumo de alimento e água também apresentou resultados semelhantes para os Grupos P e MP: todos apresentaram aumento na ingestão de água e diminuição do consumo de ração durante o protocolo. Para os testes de consumo e preferência de líquido, tanto o Grupo P quanto o MP apresentaram oscilação de preferência no período sem estressores e aumento da ingestão de sacarose durante as semanas de submissão ao protocolo, ou seja, não produziram anedonia. Os resultados permitem afirmar que o grupo que passou pelo procedimento de manipulação neonatal separados da mãe por 15min não apresentou diferença em comparação com o grupo que não foi manipulado, principalmente em relação aos testes de ingestão e preferência, quando submetidos ao CMS

Made available in DSpace on 2016-04-29T13:18:12Z (GMT). No. of bitstreams: 1 Clarissa Moreira Pereira.pdf: 1514649 bytes, checksum: b0c2f013c0bba99af8bfeae86e872f2c (MD5) Previous issue date: 2009-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Chronic Mild Stress (CMS) is an experimental model that attempts to reproduce, in laboratory, real life stressing conditions in rats, by using a stress protocol to study the effects of the exposition to these stressors in the subjects behavior. In the present experiment, the purpose was to identify if whole protocol, and not some of the stressor stimuli specifically (water and food deprivation) are responsible for producing the effects frequently described in literature (decreased sweet substance ingestion and preference and body weight when submitted to the protocol). It was also verified the influence of operant concurrent schedule sessions (water-sucrose) in all the effects considered. The results were described on (a) body weight, (b) food and water consumption, (c) sucrose ingestion and preference, and (d) concurrent schedule performance. Deprivation itself and incomplete protocol (with no deprivation conditions) produced effects on body weight, food and water consumption, and sucrose preference and ingestion. However, the two together complete protocol showed itself to be critical to produce these results. Some operant behavior features differ depending on whether the subjects are deprived or not before the session, but they don t among subjects that are submitted to the other stressors (other than deprivation). According to these results, there are two possible suggestions: (a) neonatal handling can be responsible for the inexistency of the common effects of the protocol in the subjects behavior; and (b) the continuous exposure to sucrose can have similar effects to analgesia, making it possible that effects of the protocol are not produced. One issue is considered important, considering these results: measures, other than just sucrose ingestion and preference, must be taken into account in CMS studies / O Chronic Mild Stress (CMS), ou estresse crônico moderado, é um modelo experimental que tenta reproduzir, em laboratório, através do uso de um protocolo de estressores, condições da vida real, em ratos, para estudo dos efeitos da exposição a esses estressores no comportamento dos sujeitos. No presente estudo, pretendeu-se verificar se o protocolo completo, e não apenas alguns estressores apresentados isoladamente (privação de água e privação de alimento), seriam responsáveis pelos efeitos comumente descritos na literatura (diminuição na ingestão e preferência por substância doce e no peso pela submissão ao protocolo). Foi verificada também a interferência de sessões operantes em esquema concorrente (água-sacarose) em todos os efeitos considerados. Os resultados encontrados foram analisados com relação a (a) peso corporal, (b) consumo de alimento e água, (c) ingestão de líquidos nos testes da gaiola viveiro e (d) desempenho em esquema concorrente. Tanto a privação isoladamente quanto o protocolo incompleto (sem privação) produziram efeitos no peso dos sujeitos, no consumo de alimento e água e na ingestão e preferência de líquidos. Porém, a junção de ambos o protocolo completo se mostrou crítica na produção dos resultados com relação a essas medidas. Alguns aspectos do desempenho operante diferem a depender dos sujeitos estarem privados ou não previamente às sessões, mas não diferem entre sujeitos que passam ou não pelo restante dos estressores. Com relação aos resultados, duas sugestões são colocadas: (a) a manipulação neonatal pode ser uma variável responsável pela não produção de todos os efeitos do protocolo no comportamento dos sujeitos; e (b) a exposição prolongada à sacarose pode ter efeitos similares à analgesia nos sujeitos, fazendo com que os possíveis efeitos do protocolo de estressores não sejam produzidos. É levantado um ponto considerado importante, a partir dos resultados: outras medidas, que não apenas a ingestão de líquidos, devem ser consideradas para análise em estudos com o CMS

Ο νεογνικός χειρισμός, ένα πειραματικό μοντέλο πρώιμων εμπειριών, είναι γνωστό ότι επηρεάζει τη λειτουργία του άξονα υποθαλάμου-υπόφυσης-επινεφριδίων βελτιώνοντας την προσαρμοστικότητα, την αντιμετώπιση του στρες, τις διανοητικές ικανότητες και γενικά τις διεργασίες του εγκεφάλου που σχετίζονται με την πλαστικότητα. Προηγούμενες μελέτες έδειξαν ότι ο νεογνικός χειρισμός τροποποιεί τη σεροτονινεργική, τη ντοπαμινεργική αλλά και τη γλουταμινεργική οδό, την κύρια διεγερτική οδό στον εγκέφαλο, μέσα από εκλεκτικές επιδράσεις τόσο στους υποδοχείς NMDA όσο και στους υποδοχείς AMPA στον εγκέφαλο των επίμυων. Το σύστημα των ενδοκανναβινοειδών του εγκεφάλου, δρώντας μέσω των υποδοχέων CB1, εμπλέκεται καθοριστικά στη διατήρηση της ομοιόστασης και της ενεργειακής ισορροπίας του οργανισμού, ενώ φαίνεται να έχει «αγχολυτικό» ρόλο ρυθμίζοντας συμπεριφορές όπως η πρόσληψη τροφής, ο φόβος και το άγχος, το αίσθημα ανταμοιβής και ευφορίας. Επίσης, διαδραματίζει έναν ιδιαίτερο ρόλο στην ανάπτυξη του νευρικού συστήματος καθοδηγώντας τη δημιουργία των συνδέσεων του φλοιού με τις υποφλοιώδεις δομές. Πρόκειται για ένα δυναμικό σύστημα το οποίο δύναται να τροποποιείται με την εμπειρία, ενώ η λειτουργία του μεταβάλλεται κατά τα διάφορα αναπτυξιακά στάδια, με τις κορυφαίες αλλαγές να σημειώνονται κατά την περίοδο της εφηβείας. Σκοπός της παρούσας εργασίας ήταν να ερευνηθεί εάν ο νεογνικός χειρισμός μπορεί να επιδρά στο σύστημα των ενδοκανναβινοειδών και αν η επίδραση αυτή μπορεί να παρουσιάζεται με διαφορετικό τρόπο στην εφηβεία από την ενηλικίωση. Μελετήσαμε έτσι την έκφραση των υποδοχέων CB1 στον εγκέφαλο εφήβων και ενηλίκων επίμυων, σε περιοχές που εμπλέκονται στην αντιμετώπιση του στρες, στη μνήμη, τη μάθηση, το συναίσθημα και την ανταμοιβή, όπως ο προμετωπιαίος φλοιός, η αμυγδαλή, ο ιππόκαμπος, το ραβδωτό σώμα και ο επικλινής πυρήνας. Σύμφωνα με το παρόν πρωτόκολλο νεογνικού χειρισμού, κάθε νεογνό απομακρυνόταν από τη φωλιά για 15 λεπτά καθημερινά από την πρώτη μεταγεννητική ημέρα μέχρι τον απογαλακτισμό του, τρεις εβδομάδες μετά. Χρησιμοποιήθηκε η τεχνική του υβριδισμού in situ για την εντόπιση και ποσοτικοποίηση των επιπέδων mRNA των υποδοχέων CB1 και η μέθοδος της ποσοτικής αυτοραδιογραφίας in vitro για την μέτρηση των επιπέδων δέσμευσης του υποδοχέα. Τα αποτελέσματά της έρευνας έδειξαν ότι ο νεογνικός χειρισμός προκάλεσε μεταβολές στην έκφραση των υποδοχέων CB1, με τρόπο ειδικό σε κάθε περιοχή και συχνά διαφορετικό στους εφήβους και στους ενήλικες επίμυες. Πιο συγκεκριμένα, οι έφηβοι επίμυες με χειρισμό παρουσίασαν αυξημένα, ενώ οι ενήλικες μειωμένα επίπεδα δέσμευσης των υποδοχέων CB1 στις περιοχές του προμετωπιαίου φλοιού και του επικλινούς πυρήνα. Επιπρόσθετα, ο νεογνικός χειρισμός επέφερε αύξηση τόσο των επιπέδων mRNA όσο και των επιπέδων δέσμευσης στο ραβδωτό σώμα των εφήβων επίμυων. Αντίθετα, οι ενήλικες επίμυες που είχαν υποστεί χειρισμό παρουσίασαν μείωση στα επίπεδα του mRNA στο ραβδωτό σώμα, χωρίς μεταβολή στα επίπεδα του λειτουργικού υποδοχέα στη συγκεκριμένη περιοχή. Στην αμυγδαλή, ο νεογνικός χειρισμός προκάλεσε αύξηση στα επίπεδα του mRNA ανεξάρτητα από την ηλικία, η οποία ακολουθήθηκε από αύξηση στα επίπεδα δέσμευσης του υποδοχέα CB1 μόνο στους εφήβους επίμυες με χειρισμό, ενώ οι ενήλικες δεν παρουσίασαν στατιστικώς σημαντική μεταβολή. Οι μεταβολές στην έκφραση του υποδοχέα CB1 θεωρούμε ότι συντελούνται μέσω επιγενετικής ρύθμισης ως αποτέλεσμα του νεογνικού χειρισμού και συνιστούν μια αντισταθμιστική απάντηση στη μεταβολή της ανάδρομης ενδοκανναβινοειδούς σηματοδότησης. Έτσι, το αποτέλεσμά μας στον προμετωπιαίο φλοιό, είναι πιθανόν να αντανακλά την ενίσχυση (στους ενήλικες) ή την αποδυνάμωση (στους εφήβους) της αρνητικής ανάδρομης ρύθμισης των κορτικοειδών στον άξονα του στρες, δεδομένων των αντίρροπων μεταβολών στα επίπεδα δέσμευσης του υποδοχέα CB1 στις δύο διαφορετικές ηλικίες. Τα αποτελέσματα στην αμυγδαλή, τον επικλινή πυρήνα και το ραβδωτό σώμα υποδηλώνουν αφενός μια διαφορετική «τονική» ρύθμιση του άξονα του στρες στα έφηβα ζώα με χειρισμό, και αφετέρου μια πιθανή εξασθένιση των λειτουργιών της ανταμοιβής και της δημιουργίας συνηθειών, η οποία ίσως να οδηγεί σε μειωμένη ευαισθησία στην εμφάνιση εθισμού. Συμπερασματικά, στην παρούσα μελέτη δείξαμε ότι η έκφραση των υποδοχέων CB1 μεταβάλλεται από το νεογνικό χειρισμό, ένα μοντέλο πρώιμης εμπειρίας, με τρόπο ειδικό ανά περιοχή και σε κάποιες περιπτώσεις διαφορετικό στους εφήβους από τους ενήλικες επίμυες. Αυτή η επίδραση του νεογνικού χειρισμού μπορεί να είναι ένας από τους παράγοντες που συμβάλλουν στην αυξημένη πλαστικότητα του εγκεφάλου των ζώων που έχουν υποστεί νεογνικό χειρισμό, η οποία εκδηλώνεται τόσο σε κυτταρικό όσο και στο συμπεριφορικό επίπεδο. / Neonatal handling, an experimental model of early-life experience, is known to affect the hypothalamic-pituitary-adrenal axis function, improving adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. Previous studies have shown that neonatal handling modifies the serotonergic, the dopaminergic and the glutaminergic pathway, the major excitatory pathway in the brain, through selective effects in both NMDA and AMPA receptors in rat brain. The endocannabinoid system of the brain, acting through CB1 receptors, is critically involved in maintaining homeostasis and energy body balance and it seems to have "anxiolytic" role regulating behaviors such as eating, fear and anxiety, feeling of reward and euphoria. It also plays a specific role in neural development, guiding the establishment of cortical-subcortical connections. It is a dynamic system which can be modified by experience and its function varies at different developmental stages, with the highest changes occuring during the period of adolescence. The present study addressed the question of whether neonatal handling might affect the endocannabinoid system and whether this effect is different in adolescence than adulthood. Thus, we investigated the expression of CB1 receptors in the adolescent and adult rat brain, looking in areas involved in coping with stress, learning and memory, emotion and reward, such as prefrontal cortex, amygdala, hippocampus, striatum and nucleus accumbens. According to the current neonatal handling protocol, each pup of a litter was removed from the nest for 15 min daily from the first postnatal day (PND1) until weaning (PND22). In situ hybridization and in vitro receptor autoradiography were used in order to localize and quantify CB1 receptor mRNA levels and receptor binding levels, respectively. We found that neonatal handling leads to changes in CB1 receptor expression, depending on brain region, which were different in adolescent and adult rats. More specifically, adolescent handled rats showed increased, while adults showed decreased CB1 receptor binding levels in prefrontal cortex and nucleus accumbens. In addition, neonatal handling induced an increase in both mRNA and binding levels in striatum of adolescents. However, adult handled rats had decreased mRNA levels in striatum, without a change in receptor binding levels in this region. In amygdala, neonatal handling brought an increase in mRNA levels regardless of age, which was followed by an increase in CB1 receptor binding levels only in adolescent handled rats, while adults showed no statistically significant change. The observed changes in CB1 receptor expression are thought to occur via epigenetic regulation in response to neonatal handling and may constitute a compensatory response to the change of retrograde endocannabinoid signaling. Thus, our result in the prefrontal cortex, may reflect strengthening (in adults) or weakening (in adolescents) of negative feedback on stress axis through corticosteroids, due to the opposite changes observed in CB1 receptor binding levels in the two different ages. The results in amygdala, nucleus accumbens and striatum suggest a different "tonic" regulation on the stress axis in adolescent handled animals, and also a reduced reward and habit formation function, which may lead to reduced susceptibility to addiction. In conclusion, in the present study we have shown that neonatal handling, an early-life experience model, leads to changes in CB1 receptor expression in a region- and age-specific manner. This effect of handling could be one of the factors underlying the increased blain plasticity of neonatally handled animals, which is manifested both at the cellular and at the behavioural/systemic level.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Exposure of rodents to stimuli like neonatal handling, have been described to cause behavioral and physiological benefits in adulthood. On the other hand, exposure to adults to stressful environments can result in emotional and neuropsychiatric pathologies. This study aimed to investigate the possible influence of two forms of neonatal handling as tactile stimulation (TS) and maternal separation (MS) on the emotional status of rats exposed to chronic mils stress (CMS) grouped housing, lights on overnight, damp sawdust and others. Furthermore, we aimed to evaluate if neonatal TS could modify anxiolytic effects observed with a low dose of a benzodiazepine drug in adult rats. In the first study, male Wistar pups were submitted daily to TS or MS, from postnatal day one (PND1) to PND21, for 10 min. Unhandled (UH) animails remained in next without any manipulation. In adulthood (PND67), half the animals of each group were exposed to the CMS for 3 weeks and observed in sucrose preference (SP), elevated plus-maze (EPM) and defensive burying test (DBT), followed by euthanasia for biochemical and hormonal assessments. CMS reduced SP, increased anxiety on EPM and DBT and increased adrenal weight. In addition, some parameters of antioxidant defenses in plasma, hippocampus and cortex were altered with exposure to CMS, whereas an increase in protein oxidation in hippocampus and cortex also were observed. In contrast, both forms of neonatal handling were able to prevent changes in SP, anxiety behavior on DBT and adrenal weight CMS-induced. Furthermore, they also prevented alterations in antioxidant defenses in plasma, hippocampus and cortex and protein oxidation in hippocampus. Only TS prevented CMS-induced anxiety symptoms on EPM and protein oxidation in cortex. Furthermore, TS was associated with lower levels of cortisol than in UH rats before and after CMS exposure. Since TS presented better results, we performed a second experiment only with this neonatal handling. In adulthood, the animals received a single administration of diazepam (DZP) (0.25 mg/kg body weight-i.p.) or vehicle (V) and were submitted to behavioral evaluations. DZP treatment reduced anxiety-like behaviors in EPM and increased exploration in EPM, staircase and open field tasks only in TS group. Considering UH animals, DZP treatment only increased exploration in staircase test. TS animals treated with DZP presented reduced anxiety-like behaviors in many parameters of EPM test, increased exploratory behavior in staircase and open field tasks and less immobility in DBT. The results of this study showed the protective role of neonatal handling, especially TS, which may enhance ability to cope with stressful situations in adulthood and affect the response for benzodiazepine substances during this period. / A exposição de roedores a estímulos como o manuseio neonatal, tem sido descrita por causar alterações comportamentais e fisiológicas benéficas na vida adulta. Já a exposição de animais adultos a ambientes estressantes, pode resultar em prejuízos emocionais e patologias neuro-psiquiátricas. Este estudo objetivou investigar a possível influência de dois tipos de manuseio neonatal, estimulação tátil (ET) e separação materna (SM), sobre o estado emocional de ratos após exposição ao estresse crônico e moderado (ECM) superlotação, distúrbio do ciclo claro/escuro, serragem molhada entre outros. Também objetivamos avaliar se a ET neonatal poderia modificar os efeitos ansiolíticos observados com uma baixa dose de fármaco benzodiazepínico nos animais adultos. No 1º estudo, filhotes machos de ratos Wistar foram submetidos diariamente à ET ou SM desde o dia pós-natal 1 (DPN1) até o DPN21, durante 10 minutos. Os animais não manuseados (NM) permaneceram no ninho sem nenhuma manipulação. Na vida adulta (DPN67), metade dos animais de cada grupo foram expostos ao ECM durante 3 semanas e observados no teste de preferência pela sacarose (PS), labirinto em cruz elevado (LCE) e teste defensivo de cavocar (TDC), seguido de eutanásia para avaliações bioquímicas e hormonais. O ECM reduziu a PS, aumentou a ansiedade no LCE e TDC e aumentou o peso das adrenais. Alguns parâmetros de defesas antioxidants em plasma, hipocampo e córtex foram alterados pela exposição ao ECM, enquanto um aumento da oxidação proteica em hipocampo e córtex também foram observados. Ambas as formas de manuseio neonatal foram capazes de prevenir as mudanças na PS, ansiedade no TDC e peso das adrenais. Também preveniram as alterações nas defesas antioxidants em plasma, hipocampo e córtex e a oxidação proteica no hipocampo. Apenas a ET preveniu a ansiedade induzida pelo ECM no LCE e a oxidação proteica no córtex. Além disso, a ET foi associada aos menores níveis de cortisol comparado aos ratos NM antes e após a exposição ao estresse. Uma vez que a ET apresentou melhores resultados, realizamos um 2º experimento apenas com essa forma de manuseio neonatal. Na vida adulta, os animais receberam uma única administração de diazepam (DZP) (0.25 mg/Kg peso corporal-i.p.) ou veículo (V), e foram submetidos às avaliações comportamentais. O tratamento com DZP reduziu comportamentos de ansiedade no LCE e aumentou a exploração no LCE, no teste da escada e campo aberto apenas no grupo ET. Considerando os animais NM, o tratamento com DZP apenas aumentou a exploração no teste da escada. Os animais do grupo ET tratados com DZP apresentaram menor ansiedade em vários parâmetros do LCE, maior comportamento exploratório no teste da escada e campo aberto e menor imobilidade no TDC. Os resultados do presente estudo demonstraram o papel protetor do manuseio neonatal, especialmente da ET, a qual pode melhorar a habilidade para lidar com situações estressantes na vida adulta e afetar a resposta a substâncias benzodiazepínicas nesse período.