Sabendo-se que o sistema colinérgico muscarínico modula funções cognitivas, propomos estudar as toxinas muscarínicas isoladas do veneno da M. lemniscatus, MT-Mlα e MT-Mlβ, sobre o processo de aprendizado e memória. Ratos Wistar machos foram injetados por via intrahipocampal com MT-Mlα, MT-Mlβ ou solução de Ringer (SRg). Após um período de sete dias de treino no Labirinto Aquático de Morris (LAM), os ratos receberam uma das toxinas ou SRg (dia da inoculação) e 20 min e 24 h após a inoculação foram testados no LAM. A MT-Mlα reduziu o tempo de permanência no quadrante do dia anterior, indicando interferência na evocação da memória. Por outro lado, a MT-Mlβ causou um efeito facilitatório quanto à recuperação da localização da plataforma. A ansiedade foi avaliada no Labirinto em Cruz Elevado, o treino foi feito após 30 min da injeção e o teste 24 h após. O tratamento com a MT-Mlβ mostrou ter um efeito ansiogênico, o que pode ter contribuído para o efeito facilitatório sobre a memória, pois sabe-se que a ansiedade até certo nível, pode favorecer o desempenho cognitivo. / Knowing that the muscarinic cholinergic system modulates cognitive functions, we propose to study the muscarinic toxins isolated from the venom of M. lemniscatus, MT-Mlα and MT-Mlβ, on the process of learning and memory. Male Wistar rats were injected by intrahippocampal pathway with MT-Mlα, MT-Mlβ or Ringer\'s solution (SRG). After a period of seven days training in Morris Water Maze (MWM), rats received a toxin or SRG (Inoculation day) and 20 min and 24 h after inoculation were tested in LAM. The MT-Mlα reduced the time spent in the quadrant of the previous day, indicating interference in the evocation of memory. On the other hand, the MT-Mlβ caused a facilitatory effect in recovering the location of the plataform. Anxiety was assessed in the Elevated Plus Maze, the training was done at 30 min after injection and 24 h after the test. Treatment with MT-Mlβ shown to have an anxiogenic effect, which may have contributed to the facilitatory effect on memory, since it is known that anxiety to a certain level can help cognitive performance.

Learning allows animals to adapt their behaviour to changes in the environment. In humans and other mammals, memories are stored in the hippocampus and cerebellum, whereas in insects, they are stored inside the mushroom bodies (MB). Here, MB-intrinsic Kenyon cells (KCs) form plastic synapses to MB output neurons (MBONs) that are modulated by the reinforcing action of dopaminergic neurons (DANs). Despite decades of research on the MB, the main neurotransmitter underlying the plastic KC → MBON synapse has remained a mystery. Here, I show that this synapse is cholinergic in the fruit fly Drosophila melanogaster. MBONs show fast excitatory responses to direct acetylcholine (ACh) application. KCs synthesise ACh-related proteins ChAT and VAChT. MBONs express and require nicotinic ACh receptors (nAChRs) to become fully activated by odour presentation. Lastly, artificial activation of KCs leads to MBON calcium responses that are blocked by nicotinic antagonists and genetic reduction of VAChT in KCs. Short neuropeptide F (sNPF) may play a role as a modulatory co-transmitter that can either excite or inhibit specific MBONs and DANs. The retrieval of memories is state-dependent and known to potentially change the original memory. Fruit flies need to be hungry to express appetitive memories. Hunger state depends on insulin signalling that activates the GABAergic MBON MVP2, while appetitive memory retrieval depends on decreased activity in M4/6 MBONs. Here, I show that optogenetic MVP2 activation acutely inhibits M4/6 odour responses, rendering MVP2 an inhibitory MBON interneuron. I also show that other MBONs are functionally connected to DANs, thus linking memory reinforcement and retrieval pathways in a way that enables the updating of the original memory. These findings show that associative memories in Drosophila are initially formed at cholinergic-MBON synapses, and can be retrieved and modified through an intricate KC-MBON-DAN network.

A sepse é caracterizada por um desequilíbrio entre a resposta pró- e anti-inflamatória às infecções. Um dos principais componentes da resposta do hospedeiro no choque séptico são as interações recíprocas entre o sistema imune e o sistema nervoso central, desta forma o objetivo deste estudo foi investigar o desenvolvimento de alterações neurológicas e sua associação com alterações imunológicas em fases iniciais e tardias após a sepse por ligação e perfuração cecal (CLP). Dividimos em três experimentos: agudo, crônico e efeito da ACh na evolução tardia da sepse. No experimento agudo utilizamos camundongos Balb/c, induzimos sepse por CLP em diferentes gravidades (leve, moderado e grave), 6 horas após o CLP foi realizado teste comportamental SHIRPA e logo após os animais foram sacrificados. No experimento crônico os camundongos Balb/c foram submetidos ao CLP leve, o SHIRPA foi realizado 6 horas e 15 dias após o CLP e os animais foram sacrificados 15 dias após o CLP. No experimento dos efeitos da ACh utilizamos camundongos Balb/c que receberam a droga donepezila (5 mg/kg/dia, oralmente) sete dias antes do CLP leve até o dia do sacrifício e os camundongos homozigotos mutantes VAChT KD também submetidos ao CLP leve. O teste comportamental SHIRPA foi realizado 6 horas após o CLP e os animais sacríficos 15 dias após o CLP. O plasma, o baço e o hipocampo foram removidos em todos os experimentos. Os níveis do S100? foram medidos no plasma. Os baços foram pesados, e por citometria de fluxo foi caracterizado os linfócitos (linfócitos T citotóxicos, linfócitos T auxiliares, linfócitos B, células T reguladoras e células Th17) e morte celular (Apoptose inicial, necrose e apoptose tardia). Os níveis de citocinas no baço, hipocampo e plasma foram determinados por ELISA. Nossos resultados mostram que no experimento agudo, 6 horas após o CLP a encefalopatia é diferente dependendo da gravidade da sepse, e o perfil de linfócitos no baço não é alterado por nenhuma gravidade da sepse. No entanto, a ativação de células do baço foi indicada no nosso estudo por variações na quantidade de citocinas no baço. No experimento crônico observamos que 15 dias após o CLP os animais apresentam encefalopatia séptica, e esta está correlacionada com a diferenciação e morte celular de linfócitos do baço, o que leva a um alto perfil imunossupressor. No experimento da ACh mostramos que a estimulação da transmissão colinérgica, utilizando donepezila, diminui a inflamação, por aumentar linfócitos, morte linfocitária e diminuir citocinas pró-inflamatória. E, ao contrário, a diminuição da transmissão colinérgica, experimento VAChT KD, observouse uma diminuição de linfócitos, sem morte celular e aumento da inflamação. Desta forma, concluímos que a alteração neurológica nos animais com sepse está associada com as alterações imunológicas tardias e que a ACh tem um importante papel no perfil imunológico 15 dias após o CLP / Sepsis is characterized by an imbalance between pro- and anti-inflammatory responses to infection. One of the main components of the host response in septic shock are the reciprocal interactions between the immune system and the central nervous system, so the aim of this study was to investigate the development of neurological disorders and their association with immunological changes in early and late stages after sepsis by cecal ligation and puncture (CLP). We divided in three experiments: acute, chronic and chronic ACh. In acute experiment we use Balb/c mice, induce sepsis by CLP in different severities (mild, moderate and severe), 6 hours after CLP was conducted behavioral test SHIRPA and after the animals were sacrificed. In the chronic experiment Balb/c mice were subjected to CLP mild, the SHIRPA was performed 6 hours and 15 days after CLP, and animals were sacrificed 15 days after CLP. In chronic ACh experiment use Balb/c mice that received the drug Donepezil (5 mg/kg/day, orally) seven days before the CLP mild until the day of sacrifice and use too mice homozygous mutants KD VAChT also submitted to CLP mild. The SHIRPA behavioral test was performed 6 hours after CLP and the animals were sacrificed 15 days after CLP. The plasma, spleen and hippocampus were removed in all experiments. The levels of S100? were measured in plasma. The spleens were weighed, and flow cytometry was characterized lymphocytes (cytotoxic T lymphocytes, helper T lymphocytes, B lymphocytes, regulatory T cells and Th17 cells) and cell death (apoptosis initial, necrosis and DNA fragmentation). Cytokine levels in the spleen, hippocampus and plasma were determined by ELISA. Our results show that in the acute experiment, 6 hours after CLP encephalopathy is different depending on the severity of sepsis, since the profile of the spleen lymphocytes is not changed by any severity of sepsis. However, the spleen cell activation was shown in this study by variations in the quantity of cytokines in the spleen. In the chronic experiment we observed that 15 days after CLP animals have septic encephalopathy, and this correlates with cell differentiation and the death of spleen lymphocytes, which leads to a high immunosuppressive profile. Since in the chronic ACh experiment have shown that stimulation of cholinergic transmission, using donepezil, reduces inflammation by increasing lymphocytes, lymphocyte death and decreasing proinflammatory cytokine. And, conversely, the reduction in cholinergic transmission, KD VAChT experiment, we observed a decrease of lymphocytes, and increase cell death without inflammation. Thus, we conclude that the neurological deficits in animals with sepsis is associated with immunological late changes and ACh plays an important role in the immune profile 15 days after CLP

A farmacodependência é considerada uma doença crônica e sujeita à recaídas, na qual o indivíduo perde o controle sob a utilização de determinada droga de abuso. Conforme o usuário persiste com o uso da droga, ocorrem alterações anatômicas, fisiológicas e neuroquímicas no sistema nervoso central (SNC), as quais podem culminar no desenvolvimento de um comportamento compulsivo. A neurobiologia deste processo é complexa e envolve mecanismos de plasticidade em diferentes sistemas neurotransmissores. O principal deles é o sistema mesocorticolímbico dopaminérgico, constituído por neurônios da área ventral do tegmento mesencefálico (VTA) que se projetam para o núcleo accumbens (NAc) e ao córtex pré-frontal (CPF), diretamente relacionado aos processos motivação e recompensa. Contudo, o mesmo não é suficiente para elucidar a complexidade da doença, o que levou ao entendimento da presença de outros sistemas neurotransmissores neste processo. Sabe-se que o sistema colinérgico muscarínico está diretamente envolvido em diferentes doenças neuropsiquiátricas, incluindo a farmacodependência. Além disso, os receptores colinérgicos muscarínicos (mAChRs) estão densamente presentes em regiões límbicas, onde acetilcolina e dopamina interagem por neuromodulação. Diante disto, o objetivo deste trabalho foi investigar as possíveis alterações plásticas no sistema colinérgico muscarínico resultantes de tratamentos com cocaína que mimetizaram o consumo compulsivo humano. Para tanto, foram realizados ensaios comportamentais com camundongos Swiss machos adultos em campo aberto, tratados durante um (acute binge paradigm, 30 mg/kg) ou 14 dias (escalating dose binge paradigm, 15 - 30 mg/kg) com cocaína. Os animais receberam 3 injeções intraperitoneais (i.p.) de cocaína com intervalos de 60 minutos, durante os quais a atividade locomotora foi avaliada. Após a análise comportamental, os animais foram eutanasiados por decapitação para a remoção do encéfalo e dissecação do estriado, CPF e hipocampo, regiões cerebrais cruciais para o processo fisiopatológico da farmacodependência. Componentes do sistema dopaminérgico (receptores D1 e D2) e colinérgico muscarínico (M1-M5 mAChRs, ChAT, VAChT e AChE) foram avaliados por Immunoblotting. O sangue dos animais foi coletado para a realização das dosagens de cocaína e benzoeilecgonidina por UPLC-MS/MS. O desempenho locomotor total dos animais tratados com cocaína foi superior ao dos animais controle. O grupo tratado com escalonamento de dose desenvolveu sensibilização comportamental aos efeitos psicoestimulantes da cocaína no segundo dia de tratamento e, a partir dele, a atividade locomotora total manteve a mesma magnitude. Além disso, conforme o aumento da dose, os animais mantiveram um nível de atividade superior ao basal, mesmo após o término do experimento. As análises de Immunoblotting mostraram alterações dopaminérgicas e colinérgicas. No estriado observou-se redução da densidade de D2R após o tratamento de 14 dias e aumento na densidade de M3 mAChR após o tratamento agudo. Já no hipocampo observou-se redução de D1R e aumento de D2R, M1 e M5 mAChR após o tratamento crônico; e um aumento na densidade de M3 mAChR após o tratamento agudo. No CPF, foi evidenciada redução de M3 e de M5 mAChR após o tratamento cônico de 14 dias. Em relação às moléculas colinérgicas, observou-se, após o tratamento crônico, aumento da quantidade de ChAT em todas as estruturas estudadas. Além disso, VAChT mostrou-se aumentado no hipocampo após ambos os tratamentos. As dosagens plasmáticas revelaram a presença de 20,38 ± 3,4 ng/mL de cocaína e 224,6 ± 24,02 ng/mL de benzoilcgonina (BZE) nos animais do grupo agudo e, nos do grupo crônico, 62,26 ± 10,56 ng/mL e 375,1 ± 25,62 ng/mL de cocaína e BZE respectivamente. / Drug addiction is a chronic releapsing disorder characterized by the loss of control in limiting drug intake. As the drug use persists, anatomical, physiological and neurochemical changes occur in the central nervous system (CNS), which may lead to the development of compulsive behaviors. The neurobiology of this process is complex and involves mechanisms of plasticity in different neurotransmitter systems. The main one is the mesocorticolimbic dopaminergic system, composed by neurons from the ventral tegmental area (VTA) that projects to the nucleus accumbens (NAc), which is directly related to motivation and reward processes. However, just dopamine is not enough to elucidate the complexity of the disease, leading to the comprehension of another neurotransmitters system involved. It is known that the cholinergic system is involved in different neuropsychiatric disorders, including drug addiction. Furthermore, cholinergic muscarinic receptors (mAChRs) are densely present in limbic regions, where acetylcholine and dopamine interact by neuromodulation. Considering that, the aim of this study was to evaluate the existence of neuroadaptative changes in the cholinergic muscarinic system induced by cocaine in a compulsive-like behavior model in mice. Swiss-Webster adult male mice received 3 daily injections (i.p) of cocaine or saline, with a 60-min interval among them, either acutely (acute binge paradigm) or for 14 consecutive days (escalating dose binge paradigm). The locomotor activity was monitored in the open field during 60 min, in 5 min bins, after each injection. After behavioral analysis animals were euthanized by decapitation and the brain regions of striatum, hippocampus and prefrontal cortex, involved in the pathophysiology of addiction were dissected. Dopaminergic receptors (D1R and D2R), cholinergic muscarinic receptors (M1-M5 mAChRs), choline acetylytransferase (ChAT), acetylcholine vesicular transporter and acetylcholinesterase (AChE) were quantified by Immunoblotting. Blood samples were collected with heparin and plasma was separated and stored with 2% sodium fluorite at -80ºC for cocaine and benzoilecgonine quantification by UPLC-MS/MS. In the open field, animals treated with cocaine showed an increase in locomotor activity compared to control. Cocaine induced behavioral sensitization, in the escalating dose group on day 2, and after that the locomotor activity had the same magnitude until day 14th. These animals also kept the locomotor activity elevated even after the last injection. Immunobltting shows dopaminergic and cholinergic changes. An increase in M3 was observed in both hippocampus and striatum of animals acutely treated. After 14 days, there was an increase in M1, M5 and D2 and a decrease in D1 in hippocampus. There was also a decrease in D2 in the striatum; and finally, there was a decrease in M5 and M3 in the prefrontal cortex. ChAT densities were higher in all regions after the chronic treatment. Besides that, VAChT were higher in the hippocampus after both acute and chronic treatments. UPLC-MS/MS for cocaine and benzoilecgonine demonstrated the presence of 20,38 ± 3,4 ng/mL of cocaine and 224,6 ± 24,02 ng/mL of BZE in the acute binge group; and, 62,26 ± 10,56 ng/mL and 375,1 ± 25,62 ng/mL of cocaine and BZE, respectively in the escalating dose animals.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2016-11-16T13:07:17Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Efeitos do Riluzole no Sistema Nervoso Central e Periférico de vertebrados..pdf: 3331266 bytes, checksum: 695a072e1aef84e61ff2e6ef010514a2 (MD5) / Made available in DSpace on 2016-11-16T13:07:17Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Efeitos do Riluzole no Sistema Nervoso Central e Periférico de vertebrados..pdf: 3331266 bytes, checksum: 695a072e1aef84e61ff2e6ef010514a2 (MD5) Previous issue date: 2014-05-15 / O Riluzole é quimicamente relacionado aos benzotiazóis e é conhecido como um agente neuroprotetor, possuindo propriedades anticonvulsivantes, analgésicas, anestésicas, e sedativas. A ação neuroprotetora mais conhecida desta droga ocorre através da inibição da transmissão glutamatérgica no sistema nervoso central (SNC). Nesse trabalho, o Riluzole foi ensaiado sobre a junção neuromuscular esquelética (JNM) de aves visando estudar sua interação com o sistema nervoso periférico (SNP). Também foi verificada a ação do Riluzole em fatias de hipocampo de camundongos, comparando os resultados com agentes antiinflamatórios como o extrato de Hypericum brasiliense (HBE) e seu principal composto, quercetina, frente ao veneno de serpente Crotalus durissus terrificus (Cdt). No SNP, o Riluzole foi ensaiado em preparação músculo biventer cervicis de pintainhos, em banho de órgão isolado, nas doses de 5, 10 e 20 µM. Foram obtidos registros da amplitude da força de contração muscular em presença ou ausência de Riluzole durante 120min, e as curvasresposta à adição exógena de acetilcolina (ACh – 110 µM) e ao cloreto de potássio (KCl – 20 mM), antes e após a incubação com o tratamento. O Riluzole induziu respostas tempo e dosedependentes. Na concentração de 5 µM houve uma diminuição gradativa e significativa da resposta contrátil (p<0.05). Na concentração de 10 µM, houve uma facilitação significativa (p<0.05) da resposta contrátil e das curvas evocadas pelo KCl e ACh. No entanto, na dose de 20 µM houve uma estabilização da contratilidade em relação ao controle. Em todas as doses de Riluzole ensaiadas na JNM houve um aumento significativo da atividade da enzima acetilcolinesterase (AChE). Na sequência da verificação do mecanismo de ação do Riluzole sobre a placa motora, registros eletromiograficos foram tomados na presença dos inibidores especificos, Neostigmina e d-Tubocurarina, onde foi observada uma reversão dos efeitos quando Riluzole foi adicionado ao meio. Como modelo celular de SNC, a ação do Riluzole foi ensaiada em fatias de hipocampo e a viabilidade destas frente ao veneno de Cdt foi observada por meio da atividade de desidrogenases mitocondriais. Tanto Riluzole, como o extrato da planta HBE e quercetina, aumentaram a viabilidade celular em 1h de incubação a 37˚C na presença do veneno. Quercetina foi mais efetiva do que Riluzole e HBE em neutralizar a lise celular induzida pelo veneno. Assim, estes resultados demonstram a influência do Riluzole no SNC como neuroprotetor de toxinas de veneno de serpente, possivelmente atuando como agente anti-inflamatório, e no SNP, aumentando a atividade da AChE e atuando de maneira dose-dependente sobre a placa motora. / Riluzole is chemically related to benzothiazoles and it is known as a neuroprotective agent with anticonvulsant, analgesic, anesthetic and sedative properties. The neuroprotective drug action is well established being through inhibition of glutamatergic transmission in the central nervous system (CNS). In this study, we have assayed the drug Riluzole at skeletal neuromuscular junction (NMJ) of avian, seeking its interaction with the peripheral nervous system (PNS). We also tested Riluzole in the CNS of mice by comparing its results with anti - inflammatory agents, such as extract of Hypericum brasiliense (HBE) and its main isolated compound, quercetin, against the poison of Crotalus durissus terrificus (Cdt). In the PNS, Riluzole was tested in nerve-muscle preparations in chick biventer cervicis at doses of 5, 10 and 20 μM. It was obtained recordings of the muscle twitch-tension amplitude and the contracture responses to exogenous applied acetylcholine (ACh 110 μM) and potassium chloride (KCl – 20 mM) in the presence or absence of Riluzole during 120 min. Riluzole induced time and dose-dependent responses. At concentration of 5μM there was a gradual and significant decrease in the contractile response (p<0.05). The concentration of 10μM showed a significant facilitation of the contractile response (p<0.05) and an increase in the curve responses evoked by KCl and ACh. However, at a dose of 20 μM there was a stabilization of contractility compared to control. All tested doses of Riluzole showed a significant increase in the activity of the enzyme acetylcholinesterase (AChE). The action mechanism of Riluzole on the endplate was further analysed through the use of specific inhibitors, Neostigmine and dTubocurarine, a reversal of effects those was seen when Riluzole was added to the medium. As a cellular model of CNS, the action of Riluzole was tested in mice hippocampal slices. The cell viability against Cdt venom was observed through the activity of mitochondrial dehydrogenases. Riluzole as much as the plant extract HBE and its active ingredient, quercetin, increased cell viability in 1h incubation at 37˚C in the presence of the poison. However, quercetin showed to be more effective than Riluzole and HBE in neutralizing cell lysis induced by the venom. Thus, these results demonstrate the influence of Riluzole on the CNS, it showed a neuroprotective effect against snake venom toxins, possibly acting as an anti-inflammatory agent. As for the cholinergic system in the NMJ, Riluzole showed an interesting effect by increasing the activity of AChE and by acting in a dose-dependent manner over the endplate.

A destruição crônica do aparelho de inserção periodontal devido a uma intensa resposta inflamatória a bactérias conduz a uma condição clínica conhecida como doença periodontal. A inflamação periodontal geralmente provoca úlceras superficiais no sulco gengival, onde os capilares sanguíneos estão expostos a bactérias. Patógenos periodontais, assim como seus produtos podem ser translocados e liberados do sulco gengival para a corrente sanguínea e, portanto, ter efeitos periféricos. A bolsa periodontal é também importante reservatório de mediadores inflamatórios que podem atingir a corrente sanguínea. Diversos estudos avaliam a relação entre a doença periodontal e as doenças cardiovasculares. Sabe-se que muitas doenças cardiovasculares têm início em desordens vasculares, principalmente na camada endotelial dos vasos sanguíneos de grande condutância. Assim, o foco deste estudo foi investigar a reatividade vascular de aortas de animais com periodontite. Tanto a contração para a norepinefrina quanto o relaxamento para a acetilcolina estão diminuídos em animais com periodontite, mas os mecanismos para estes efeitos são diferentes. A diminuição da resposta contrátil à norepinefrina parece estar associada a um aumento da produção de óxido nítrico através da isoforma induzível da óxido nítrico sintase e a diminuição da resposta relaxante à acetilcolina parece estar associada a produção de prostanóides principalmente derivados da isoforma do tipo II da enzima ciclooxigenase. / The chronic destruction of the periodontal attachment apparatus due to an intense inflammatory response to bacteria leads to a condition known as periodontal disease. The periodontal inflammation usually causes superficial ulcers in the gingival sulcus, where blood capillaries are exposed to bacteria. Periodontal pathogens, as well as their products can be translocated and released into the gingival sulcus, subsequently into the bloodstream and thus have peripheral effects. The periodontal pocket is also an important reservoir of inflammatory mediators that can reach the bloodstream. Several studies have evaluated the relationship between periodontal disease and cardiovascular disease. It is known that many cardiac diseases begin as vascular disorders, especially in the endothelial layer of blood vessels of large conductance. Thus, the focus of this study was to investigate the vascular reactivity of aortas from animals with periodontitis. Both norepinephrine-mediated contraction and acetylcholine-mediated relaxation are decreased in animals with periodontitis, but the mechanisms are different for these responsess. The contraction appears to be associated with increased production of nitric oxide by inducible isoform of nitric oxide synthase and relaxation with prostanoids primarily derived type II isoform of the enzyme cyclooxygenase.

While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms in the hands and feet, such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our collaborators in the laboratory of Dr. M. Imad Damaj have shown that nicotine, a nicotinic acetylcholine receptor (nAChR) agonist, and R-47, an α7 nAChR silent agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy in mice. With regard to cancer, this work demonstrates that nicotine and R-47 do not enhance A549 and H460 human non-small cell lung cancer cell viability, colony formation, or proliferation alone, and they do not attenuate paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, nicotine and R-47 do not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that targeting nAChRs may be a safe and efficacious approach for the prevention and treatment of CIPN in cancer patients.

Traditionally, working memory and its neural underpinnings have been studied in the visual domain. A rich and diverse amount of research has investigated the lateral prefrontal cortex (lPFC) as a primary area for visual working memory, while another line of research has found the neurotransmitter acetylcholine (ACh) to be involved. This dissertation used auditory cues and found similar patterns of activity for processing auditory working memory information within a task compared to visual working memory processes. The first two experimental chapters demonstrated that the cholinergic system is involved in auditory working memory in a comparable fashion to its role in visual working memory. In chapter 2, blocking ACh impaired performance on an auditory working memory task in a dose dependent manner. Chapter 3 investigated the specificity of the effect of blocking ACh by administering an ACh agonist (physostigmine) at the same time as an ACh antagonist (scopolamine). When both drugs were administered together performance on the delayed matching-to-sample task (DMTS) task improved compared to performance on scopolamine alone. These results support the hypothesis that ACh is involved in auditory working memory. Chapter 4 investigated the neural correlates of auditory working memory in area 46 and found that this region of the lPFC contains neurons that are responsive to auditory working memory components in a very similar way to how it this region encodes information during visual working memory tasks. Neurons in the lPFC are responsive to visual or auditory cues, the delay portion of tasks, the wait time (i.e. decision making period), response, and reward times. This type of coding provides support for the theories that position the lPFC as a key player in recognition and working memory regardless of modality.

Doctor of Philosophy(PhD) / This thesis addresses the neurochemistry and function of specific nuclei in the autonomic nervous system that are crucial mediators of cardiorespiratory regulation. The primary aim is to build on previous knowledge about muscarinic cholinergic mechanisms within cardiorespiratory nuclei located in the ventrolateral medulla oblongata. The general focus is characterisation of gene expression patterns of specific muscarinic receptor subtypes in central nuclei involved in blood pressure control and respiratory control in normal rats. The findings were subsequently extended by characterisation of muscarinic receptor gene expression patterns in 1) a rat model of abnormal blood pressure control (hypertension) (Chapter 3) 2) a rat model of cholinergic sensitivity (Chapter 5) 3) the rat ventral respiratory group (Chapter 6) The results of a series of related investigations that ensued from the initial aims more finely characterise the neurocircuitry of the ventrolateral medulla, from a specifically cholinoceptive approach. All five muscarinic receptor subtypes are globally expressed in the ventrolateral medulla but only the M2R mRNA was significantly elevated in the VLM of hypertensive animals compared to their normotensive controls and in the VLM of animals displaying cholinergic hypersensitivity compared to their resistant controls. Surprisingly, M2R mRNA is absent in catecholaminergic cell groups but abundant in certain respiratory nuclei. Two smaller projects involving gene expression of other neurotransmitter / neuromodulators expressed in cardiorespiratory nuclei were also completed during my candidature. Firstly, the neurochemical characterisation of enkephalinergic neurons in the RVLM, and their relationship with bulbospinal, catecholaminergic neurons in hypertensive compared to normotensive animals was carried out (Chapter 4). A substantial proportion of sympathoexcitatory neurons located in the RVLM were enkephalinergic in nature. However, there was no significant difference in preproenkephalin expression in the RVLM in hypertensive compared to normotensive animals. Secondly, the identification and distribution of components of the renin-angiotensin aldosterone system (RAAS) within the brainstem, and differences in gene expression levels between hypertensive and normotensive animals was also investigated. The RAAS data was not included in this thesis, since the topic digresses substantially from other chapters and since it is published (Kumar et al., 2006). The mRNA expression aldosterone synthase, mineralocorticoid receptor (MR1), 12-lipoxygenase (12-LO), serum- and glucocorticoid- inducible kinase and K-ras) were found to be present at all rostrocaudal levels of the ventrolateral medulla. Expression of MR1 mRNA was lower in the RVLM of SHR compared with WKY rats and 12-LO mRNA levels were lower in the CVLM in SHR compared with WKY rats. Otherwise, there was no difference in gene expression level, or the method of detection was not sensitive enough to detect differences in low copy transcripts between hypertensive and normotensive animals.

Rosacea is a chronic skin disorder, characterized by redness and flushing of the cheeks, nose, chin or forehead. It has been proposed that rosacea is a result of frequent blushing (Miller, 1921; Klaber & Whittkower, 1939). However, the relationship between rosacea and blushing is uncertain. The aim of the present research was to investigate the relationship between psychological stress and vascular disturbances in rosacea. Five studies were conducted. The first study explored the relationship between rosacea and mental health while the next two investigated vascular responses in rosacea sufferers and controls to acetylcholine (which induces endothelial vasodilatation and axon reflexes) and psychological stress (embarrassment). The fourth study aimed to examine the relationship between psychological indicators and rosacea symptoms on a daily basis. The fifth study consisted of three case studies looking at the use of Cognitive Behavioural Therapy (CBT) and Task Concentration Training (TCT) with rosacea sufferers presenting with social anxiety and fear of blushing symptoms. In study 1, sixty-two participants were asked to complete the Blushing Propensity Scale (BPS), Fear of Negative Evaluation (FNE), Depression, Anxiety and Stress Scale (DASS), Social Interaction Anxiety Scale (SIAS) and Social Phobia Scale (SPS). Outcomes from the first study indicated that Type 2 rosacea sufferers (n= 12) perceived themselves as blushing more frequently and intensely than Type 1 rosacea sufferers (n=19) or controls (n=31). This suggested that Type 2 rosacea sufferers experiencing frequent blushing may have a lower sensitivity threshold to blushing episodes. In addition, Type 2 rosacea sufferers perceived themselves as more stressed than Type 1 rosacea sufferers or controls, possibly indicating that managing the condition can be stressful. Contrary to previous reports (Gupta et al., 2006; National Rosacea Society, 2005) severity of rosacea was not associated with depression, social anxiety or fear of negative evaluation. However, a few participants who reported high social anxiety and stress scores were offered psychological intervention (Study 5). The aim of the second study was to investigate vascular responses in rosacea sufferers. Cutaneous endothelial and axon reflex function was assessed using an acetylcholine dose response curve. The axon reflex was assessed by inducing a flare with ACh iontophoresis. Outcomes from this study indicated that Type 2 rosacea sufferers had a greater axon reflex response than Type 1 rosacea sufferers. Thus over-reactivity of the axon reflex in Type 2 rosacea sufferers might contribute to prolonged vasodilatation. However, cutaneous endothelial responses to ACh were similar in rosacea and control groups. The results suggested that neural pathways mediated the flushing response rather than cutaneous endothelial function. The third study investigated facial blood flow while participants attempted laboratory induced embarrassment tasks. Type 2 rosacea sufferers were found to have a greater blood flow in the facial region than Type 1 rosacea sufferers during singing and speech tasks, suggesting that Type 2 rosacea sufferers blushed more than type 1 rosacea sufferers or controls. Furthermore, Type 2 rosacea sufferers reported higher embarrassment and blushing ratings than Type 1 rosacea sufferers. This indicated that Type 2 rosacea sufferers perceived themselves as emotionally more aroused than other participants. Taken together, it would appear that a combination of physiological and cognitive factors increased facial blood flow in Type 2 rosacea sufferers in laboratory induced embarrassment tasks. The fourth study explored the relationship between stress and symptoms of rosacea. Using a diary, 15 rosacea sufferers recorded their stress, anxiety and mood and their intensity of rosacea symptoms daily. Stress was associated with increased stinging/facial redness on the same day for 1 to 2 months. Furthermore, it was associated with increased stinging ratings the next day. However, feeling anxious or having low mood was not related to increase stinging the next day. The presence of increased stress found in rosacea participants on the day where stinging and redness occurred should be taken into consideration when formulating psychological interventions for rosacea sufferers. In study 5, individual psychological intervention was provided to three participants experiencing stress, fear of blushing and social anxiety symptoms. Cognitive Behavioural Therapy (CBT) and Task Concentration Training (TCT) were helpful in managing stress, anxiety and fear of blushing symptoms in individual rosacea sufferers. Encouragingly, all participants reported a gain in their repertoire of strategies and showed a decrease in anxiety symptoms on assessment questionnaires following their intervention. Replication of the intervention protocol and investigation of other psychological approaches are required to establish best practise outcome for rosacea sufferers who require psychological interventions. The present findings suggest that over-reactivity of axon reflexes contributes to facial flushing. In addition, emotional flushing in rosacea sufferers appears to be maintained by a combination of cognitive and physiological factors. On a clinical level, the study recommends that emotional stress associated with facial flushing in rosacea sufferers to be targeted for psychological intervention.