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Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Die hohe Dichte und breite Verteilung von P2Y-Rezeptoren im Hirn von Säugetieren lässt für diese Rezeptoren eine wichtige Rolle in den Prozessen des zentralen Nervensystems vermuten. Um die Beteiligung von P2Y-Rezeptoren bei der Verarbeitung von Angst zu untersuchen, wurde in dieser Arbeit das P2Y1,11,12,13 -rezeptorspezifische ADP-Analogon Adenosin-5\'-O-2-thiodiphosphat (ADPßS), der P2X1,3-Rezeptoragonist a,b-methylen-ATP (a,bmeATP), der unspezifische P2-Rezeptorantagonist Pyridoxalphosphat-6-azophenyl-2’,4’-disulfonat (PPADS) und der spezifische P2Y1-Rezeptorantagonist N6-methyl-2’-deoxyadenosin-3’:5’bisphosphat (MRS 2179) Ratten intracerebroventrikulär injiziert und die Wirkung in einem Verhaltensversuch im standardisierten Angstmodell des elevated plus-maze untersucht. Die Substanzen wurden zu 0,5 μl verabreicht. ADPßS (50 fmol und 500 fmol) zeigte dabei anxiolytische Wirkung mit vermehrten Eintritten und gesteigerter Aufenthaltszeit der Tiere auf den offenen Armen. Eine Prämedikation mit PPADS (5 pmol) oder MRS 2179 (5 pmol) konnte diesen Effekt vollständig antagonisieren. Auch eine Vorbehandlung durch den unspezifischen NO-Synthase-Inhibitor Nw-nitro-L-arginin-methyl-ester (L-NAME) konnte die ADPβS-Wirkung verhindern. Bei alleiniger Gabe zeigten diese drei Substanzen anxiogene Wirkung mit einer verminderten Aufenthaltszeit und einer geringeren Zahl von Eintritten in die offenen Armen. Der anxiogene Effekt konnte wiederum durch eine Gabe von L-Arginin (500 pmol), einem Substrat der NO-Synthase (NOS), verhindert werden, nicht aber durch das Enantiomer D-Arginin (500 pmol), das kein Substrat der NOS darstellt. Die doppelte Immunfluoreszenz konnte die Präsenz der P2Y1-Rezeptoren an Neuronen in dorsomedialen Hypothalamus, Amygdala, Hippokampus und zentralen Höhlengrau wie auch die Kolokalisation von P2Y1-Rezeptoren und nNOS nachweisen. Die höchste Dichte an Immunoreaktivität fand sich im dorsomedialen Hypothalamus. Durch die lokale bilaterale Mikroinjektion von ADPßS und MRS 2179 konnten die in den vorausgegangen Versuchen erreichten Ergebnisse reproduziert und bestätigt werden. Zusammenfassend lässt sich postulieren, dass P2Y1-Rezeptoren maßgeblich an der Verarbeitung von Angst bei männlichen Wistar-Ratten beteiligt sind, die Wirkung eng mit der Veränderung der NO-Konzentration verbunden ist und dass diese im dorsomedialen Hypothalamus vermittelt wird. Inwieweit diese Mechanismen auch in Amygdala und Hippokampus eine Rolle spielen kann mit den vorliegenden Daten nicht abschließend beantwortet werden.

Anxiety disorders commonly emerge during adolescence, and girls are diagnosed with these disorders more frequently than boys. Understanding why anxiety disorders emerge and why non-clinical anxiety symptoms increase during adolescence is important for understanding this sex difference and how to treat adolescent sufferers. Potential mechanisms, such as puberty or cognitive biases, can be investigated both in humans and in rodent models of anxiety. This thesis aimed to characterise sex differences and changes in anxiety-like behaviour across adolescence and into adulthood in the rat, and to examine anxiety and interpretive bias in adolescent humans. In rats, we examined performance on common tests of anxiety-like behaviour: the emergence test, open field and elevated plus-maze. Exploration on these tests increased from mid-adolescence into adulthood, and greater exploration by females was apparent from late adolescence. While the behaviours themselves remain interesting, caution on interpreting these behaviours as anxiety-related warranted and is discussed throughout the thesis. Potential effects of the ovarian cycle and testing order on EPM performance were also examined. In humans, 12-14 year old adolescents complete visual and written interpretive bias tasks, this bias being considered to be a cognitive vulnerability for anxiety. The results showed that, when imagining themselves in ambiguous scenarios, girls were more negative in their interpretations than boys. Additionally, both sexes also interpreted social scenarios more negatively than non-social scenarios. As puberty is thought to be important to the emergence of disorder during adolescence, interpretive bias could potentially mediate the puberty-anxiety relationship. While more interpretive bias work is needed in both species, the recent development of interpretive bias tasks for rodents provides an opportunity to move away from difficult to interpret tests of anxiety-like behaviour in rodents, and should allow for greater convergence of the human and rodent anxiety research.

O labirinto em cruz elevado é muito usado em estudos relacionados à ansiedade em ratos. As medidas tradicionalmente usadas são o número de entradas e o tempo passado nos braços abertos. Trabalhos recentes analisam a movimentação no interior dos braços, mas não propõem um índice que resuma as análises feitas. Esta tese sintetiza as informações de um destes trabalhos em índices. Um dos índices propostos usa os tempos médios de primeira visita a cada posição do labirinto e o outro se baseia na distribuição estacionária de probabilidade, o primeiro é capaz de diferenciar grupos de ratos submetidos a diferentes drogas ansiolíticas. Além disso, a tese propõe um modelo de processo Markoviano que incorpora informações desconsideradas no modelo anterior. A comparação entre os modelos revelou valores super ou subestimados no primeiro. Por fim, esta tese propõe um modelo de cadeias de Markov considerando como estados o seguinte conjunto de comportamentos: \"rearing\", \"stretching\", \"dipping\", \"freezing\" e \"grooming\". Tal abordagem inédita, apesar de simplificar exageradamente o modelo, foi capaz de reproduzir várias características conhecidas da exploração. / The elevated plus maze is widely used in studies related to anxiety in rats. The most widely used measures are the amount of entries and time spent on open arms. Recent studies analyze the movement inside the arms, but do not propose an index that summarizes the analyzes. This thesis summarizes by indices the information in one of these studies. An index uses the first visit average time to each position of the maze and another is based on the stationary probability distribution, the first one are able to differentiate groups of rats submitted to different anxiolytic drugs. These thesis also proposes a Markov process model that incorporates more information than the other model. The comparison between the models showed over- or underestimated values in the first. Finally, this thesis proposes a Markov chains model considering the following behaviors as states: rearing, stretching, dipping, freezing and grooming. This new approach, though oversimplify the model was able to reproduce several known features of exploitation.

Há relatos de que doses baixas de pentilenotetrazol (PTZ) apresentam um efeito ansiogênico em testes comportamentais que medem ansiedade. No labirinto em cruz elevado a droga reduz a porcentagem de entradas e o tempo gasto nos braços abertos. O clordiazepóxido (CDP), por outro lado, é uma droga que tem efeitos ansiolíticos, aumentando a exploração dos braços abertos do labirinto ao reduzir a aversão natural dos ratos aos braços abertos. Obtém-se um efeito similar quando o teste ocorre no escuro. Cafeína (CAF) é um composto estimulante que, aplicada em baixas doses, estimula a atividade motora no labirinto. O presente trabalho investiga o efeito de drogas gabaérgicas nas medidas de ansiedade obtidas no labirinto em cruz elevado, na presença ou ausência de luz. Ratos distribuídos aleatoriamente em 18 grupos receberam injeções de PTZ (0, 10 e 20 mg/kg), 5 min antes do teste, CDP (0,1.5 e 3 mg/kg) ou CAF (0, 10 e 30 mg/kg) 30 min antes do teste e foram colocados no labirinto em cruz elevado por 5 minutos, permitindo sua livre exploração, sob duas condições de luminosidade: claro (22 lux) ou escuro (0 lux). Os resultados mostraram que nem o PTZ nem o CDP tiveram qualquer efeito quando os animais eram testados no escuro. No claro, o PTZ diminuiu a exploração dos braços abertos, o que foi interpretado como um efeito ansiogênico, e o CDP apresentou um efeito oposto. A CAF provocou um aumento na exploração dos braços abertos somente no escuro. Esses resultados sugerem que a luminosidade deflagra as respostas responsáveis pela esquiva dos braços abertos, e as drogas simplesmente aumentam ou bloqueiam esse efeito. Da mesma forma, a falta da aversão desencadeada pela luz permitiu que a atividade locomotora aumentasse sob ação da CAF. / Pentylenetetrazol (PTZ), in low doses, was reported to have an anxiogenic effect in behavioral tests measuring anxiety. In the elevated plus-maze, it reduces the percentage of entries into and the time spent in the open arms. Chlordiazepoxide (CDP), on the other hand, is an anxiolytic drug which increases open arm exploration in the elevated plus-maze by reducing the natural aversion of rats to the open arms. A similar effect is obtained by testing rats in the dark. Caffeine (CAF) is a stimulant compound that, when administered in low doses, stimulates locomotor activity in the plus-maze. The present work investigated the effect of gabaergic drugs on anxiety levels in the presence and absence of light. Rats were randomly divided into 18 groups and either injected 5 min before testing with PTZ (0, 10, 20mg/kg) or 30 min before testing with CDP (0, 1.5, 3.0 mg/kg) or CAF (0, 10 and 30 mg/kg) and allowed to freely explore an elevated plus-maze for 5 min under two illumination conditions: light (22 lux) or dark (0 lux). Results show that neither PTZ nor CDP had any effect when the animals were tested in the dark. When tested in the light, PTZ decreased exploratory behavior in the open arms, which is usually interpreted as an anxiogenic effect, while CDP had the opposite effect. CAF provoked an increase in open arm exploration but only when the rats were tested in the dark. These results suggest that light triggers the responses responsible for the avoidance of the open arms, and the drugs simply enhance or block them. Likewise, lacking the aversion triggered by light allowed locomotor activity to increase under the action of CAF.

The anterior claustrum (CLA) has been implicated in epileptogenesis and epileptiform activity due to its abundant and widespread bilateral connections to some of the structures believed to play an important role in seizure generalization: the motor cortex, entorhinal cortex, limbic structures, and brainstem sites. Kindling in the CLA has been characterized as comprising two distinct phases: an early phase and a late phase. Early phase seizures progress quickly into generalized seizures, are short in duration, and resemble cortical seizures. Late phase seizures are characterized as being more severe in intensity, having longer durations, and resembling limbic-type seizures.<p>It is unknown whether kindling in the CLA will lead to changes in behaviour as seen after kindling of limbic sites. Thus, I measured the behavioural effects of kindling in the anterior CLA to investigate potential changes in learning, memory, and anxiety-related behaviours. I hypothesized that changes in behaviour would occur after kindling of late phase seizures, because of their close resemblance to limbic-type seizures, but not after kindling of early phase seizures. Anxiety-like behaviour was assessed in the elevated plus maze and open field. Object memory was assessed in an object recognition test, and spatial learning and memory were assessed in the water maze.<p>I found no significant changes in behaviour in the late phase group in comparison to the early phase and control groups. Thus, contrary to my hypothesis, late phase kindling of the CLA does not produce changes in learning and memory or alterations in anxiety-related behaviours.

The anterior claustrum (CLA) has been implicated in epileptogenesis and epileptiform activity due to its abundant and widespread bilateral connections to some of the structures believed to play an important role in seizure generalization: the motor cortex, entorhinal cortex, limbic structures, and brainstem sites. Kindling in the CLA has been characterized as comprising two distinct phases: an early phase and a late phase. Early phase seizures progress quickly into generalized seizures, are short in duration, and resemble cortical seizures. Late phase seizures are characterized as being more severe in intensity, having longer durations, and resembling limbic-type seizures.<p>It is unknown whether kindling in the CLA will lead to changes in behaviour as seen after kindling of limbic sites. Thus, I measured the behavioural effects of kindling in the anterior CLA to investigate potential changes in learning, memory, and anxiety-related behaviours. I hypothesized that changes in behaviour would occur after kindling of late phase seizures, because of their close resemblance to limbic-type seizures, but not after kindling of early phase seizures. Anxiety-like behaviour was assessed in the elevated plus maze and open field. Object memory was assessed in an object recognition test, and spatial learning and memory were assessed in the water maze.<p>I found no significant changes in behaviour in the late phase group in comparison to the early phase and control groups. Thus, contrary to my hypothesis, late phase kindling of the CLA does not produce changes in learning and memory or alterations in anxiety-related behaviours.

No description available.