Eventuais alterações no substrato anatômico miocárdico, no sistema nervoso autônomo (SNA) cardíaco intrínseco, envolvendo os plexos ganglionares (PG) comumente presentes em organizações de gordura epicárdicas denominadas fatpads (FP) ou a expressão dos receptores muscarínicos, poderiam ser responsáveis pela gênese e manutenção da fibrilação atrial (FA). Com o objetivo de analisar a relação entre fibrilação atrial permanente (FAP) e possíveis alterações anatômicas e micromorfológicas do coração, do SNA cardíaco intrínseco e da expressão dos receptores muscarínicos miocárdicos, foram estudados 13 corações de autópsias de portadores de FAP e cardiopatia crônica definida (grupo I) e 13 casos pareados pela mesma doença cardíaca, porém sem esta arritmia (grupo II). Foram analisados a anatomia da drenagem venosa do átrio esquerdo (AE), peso do coração, espessura do septo ventricular e diâmetro dos FP epicárdicos. Foram ressecadas duas amostras no átrio direito (AD1 e AD2), três no átrio esquerdo - no trajeto médio da VoAe (AE1), na junção da veia pulmonar superior esquerda (AE2) e na aurícula (AE3), três em FPs, atrial esquerdo superior (FP1), atrial direito posterior (FP 2) e no atrial esquerdo póstero-medial (FP 3) e uma amostra do septo ventricular (SIV), como controle. As alterações estruturais das fibras miocárdicas, as espessuras do epicárdio, endocárdio e miocárdio e o percentual de colágeno intersticial no miocárdio foram analisados através de histomorfometria computadorizada sob coloração de tricrômio de Masson. O SNA cardíaco intrínseco foi analisado através imuno-histoquímica para S-100 e tirosina-hidroxilase quanto a: quantidade e área das fibras nervosas, quantidade e área média de fibras simpáticas, quantidade e área média de fibras parassimpáticas e proporção de fibras simpáticas/parassimpáticas. A expressão miocárdica dos receptores muscarínicos 1 a 5 (M1 a M5) foi avaliada pela proporção positiva no miocárdio nos cortes AD1, AE1, AE2 e FP1. Não houve diferenças entre os grupos quanto às variáveis anatômicas e ao percentual de colágeno intersticial. A análise do SNA revelou fibras nervosas com menor área no grupo I, redução do número de fibras nervosas totais e parassimpáticas nos cortes AD1 e SIV, aumento de fibras totais e parassimpáticas AE2 e FP2 e aumento do número de fibras simpáticas nos cortes AD2, AE1, AE2 e AE3. Quanto à expressão dos receptores muscarínicos, houve aumento significante na porcentagem positiva para M1 em todas as regiões, exceto na AE1 (média de todos os cortes, grupo I 5,84 e grupo II 2,92, p=0,002); o M2 e M3 apenas junto ao FP1 (M2 grupo I 5,67 e grupo II 3,63, p=0,037; M3 grupo I 30,95 e grupo II 20,13, p=0,026) e o M4 foi aumentado no grupo I na região AE1 (grupo I 9,90 e grupo II 4,45, p=0,023); não houve alteração estatisticamente significante no M5. A anatomia e a disposição das fibras musculares atriais, bem como a fibrose intersticial não parecem estar relacionadas à FAP nos grupos estudados. Alterações no número de fibras nervosas bem como e alterações na expressão dos receptores muscarínicos atriais, especialmente o M1, particularmente em regiões próximas aos PG, parecem estar relacionadas à FAP, indicando a importância da modulação autonômica nesta arritmia / Possible changes in myocardial substrate, in the intrinsic cardiac autonomic nervous system (ANS), involving the ganglionated plexus (GP) present in fat-pads (FP) or the expression of muscarinic receptors could be responsible for the genesis and maintenance of atrial fibrillation (AF). Aiming to analyze the relationship between permanent atrial fibrillation (pAF) and possible anatomical and micromorphological heart changes, intrinsic cardiac ANS and expression of myocardial muscarinic receptors, 13 hearts from autopsies of patients with PAF and chronic heart disease (group I) were studied; and 13 cases matched by the same heart disease, but without this arrhythmia (group II). It was analyzed the anatomy of the venous drainage of the left atrium (LA), heart weight, ventricular septal thickness and diameter of epicardial FP. Two samples were taken in the right atrium (RA1 and RA2), three in the left atrium - in the middle portion of the left atrium oblique vein (LaOv LA1), at the junction of left superior pulmonary vein (LA2) and in the auricle (LA3), three FPs, left atrial superior (FP 1), right atrial posterior (FP 2) and the left atrial posteromedial (FP 3) and one sample of the ventricular septum (VS), as control. The structural changes of the myocardial fibers, thickness of the epicardium, endocardium and myocardium, and the percentage of interstitial collagen in the myocardium were analyzed by computerized histomorphometry on Masson trichrome staining. The intrinsic cardiac ANS was analyzed through immunohistochemistry for S-100 and tyrosine hydroxylase regarding the: amount and area of nerve fibers, amount and average area of sympathetic fibers, number and average area of parasympathetic fibers and sympathetic/parasympathetic fiber proportion. The myocardial expression of muscarinic receptors 1-5 (M1 to M5) was evaluated by positive ratio in the myocardium in sections RA1, LA1, LA2 and FP1. There were no differences between groups regarding the anatomical variant and the percentage of interstitial collagen. Analysis of the ANS revealed nerve fibers with the smallest area in group I, reduction in the number of total and parasympathetic nerve fibers of sections RA1 and VS, increase of total and parasympathetic fibers LA2 and FP2 and increased numbers of sympathetic fibers in sections RA2, LA1, LA2 and LA3. Regarding the expression of muscarinic receptors, there was a significantly increase in the positive percentage for M1 in all regions except for LA1 (average of all the sections, group I 5.84 and group II 2.92, p = 0.002), M2 and M3 just adjacent to the FP1 (M2 Group I 5.67 and Group II 3.63, p = 0.037; M3 Group I 30.95 and Group II 20.13, p = 0.026) and the M4 was increased in group I in the region LA1 (group I 9.90 and group II 4.45, p = 0.023) and there was no statistically significant change in the M5. The anatomy and arrangement of atrial muscle fibers, as well as the interstitial fibrosis did not appear to be related to PAF in both studied groups. Changes in the number of nerve fibers as well as changes in expression of atrial muscarinic receptors, specially the M1, particularly in regions close to the GP appear to be related to pAF, indicating the importance of autonomic modulation in this arrhythmia

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.

Locomotion is an innate behaviour that is controlled by different areas of the central nervous system, which allow for effectiveness of movement. The spinal cord is an important centre involved in the generation and maintenance of rhythmic patterns of locomotor activity such as walking and running. Interneurons throughout the ventral horn of the spinal cord form the locomotor central pattern generator (CPG) circuit, which produces rhythmic activity responsible for hindlimb movement. Motoneurons within the lumbar region of the spinal cord innervate the leg muscles to convey rhythmic CPG output to drive appropriate muscle contractions. Intrinsic modulators, such as acetylcholine acting via M2 and M3 muscarinic receptors, regulate CPG circuitry to allow for flexibility of motor output. Using electrophysiology and genetic techniques, this work characterized the receptors involved in cholinergic modulation of locomotor networks and the role and mechanism of action of a subpopulation of genetically identified cholinergic interneurons in the lumbar region of the neonatal mouse spinal cord. Firstly, the effects of M2 and M3 muscarinic receptors on the output of the lumbar locomotor network were characterised. Experiments in which fictive locomotor output was recorded from the ventral roots of isolated spinal cord preparations revealed that M3 muscarinic receptors are important in stabilizing the locomotor rhythm while M2 muscarinic receptor activation seems to increase the irregularity of the locomotor frequency whilst increasing the strength of the motor output. This work then explored the cellular mechanisms through which M2 and M3 muscarinic receptors modulate motoneuron output. M2 and M3 receptor activation exhibited contrasting effects on motoneuron function suggesting that there is a fine balance between the activation of these two receptor subtypes. M2 receptor activation induces an outward current and decreases synaptic drive to motoneurons while M3 receptors are responsible for an inward current and increase in synaptic inputs to motoneurons. Despite the different effects of M2 and M3 receptor activation on synaptic drive and subthreshold properties of MNs, both M2 and M3 receptors are required for muscarine-induced increase in motoneuron output. CPG networks therefore appear to be subject to balanced cholinergic modulation mediated by M2 and M3 receptors, with the M2 subtype also being important for regulating the intensity of motor output. Next, using Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology, the impact of the activation or inhibition of a genetically identified group of cholinergic spinal interneurons that express the Paired-like homeodomain 2 (Pitx2) transcription factor was explored. Stimulation of these interneurons increased motoneuron output through the activation of M2 muscarinic receptors and subsequent modulation of Kv2.1 channels. Inhibition of Pitx2+ interneurons during fictive locomotion decreased the amplitude of locomotor bursting. Genetic ablation of these cells confirmed that Pitx2+ interneurons increase the strength of locomotor output by activating M2 muscarinic receptors. Overall, this work provides new insights into the receptors and mechanisms involved in intraspinal cholinergic modulation. Furthermore, this study provides direct evidence of the mechanism through which Pitx2+ interneurons regulate motor output. This work is not only important for advancing understanding of locomotor networks that control hindlimb locomotion, but also for dysfunction and diseases where the cholinergic system is impaired such as Spinal Cord Injury and Amyotrophic Lateral Sclerosis.

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.

The signaling mechanisms for muscarinic receptor-mediated vasoconstriction in coronary resistance arteries were studied in KCl-arrested isolated rat hearts perfused at a constant flow rate. The cholinergic agonists acetylcholine and bethanechol were given by bolus injection or constant infusion. The coronary vascular resistance was monitored by measuring the changes in perfusion pressure. The selective muscarinic agonist bethanechol caused a similar vasoconstrictor response as ACh, but with less potency and efficacy. Bolus injection of bethanechol evoked a phasic vasoconstriction in a dose-dependent manner, while infusion of bethanechol evoked a tonic vasoconstriction without producing tachyphylaxis. Coronary vascular responses to bethanechol were further examined in the absence and presence of a specific inhibitor or blocker for the potential signaling components. The bethanechol-induced phasic vasoconstriction was eliminated by perfusion with a Ca2+ -free medium. The maximal vasoconstriction to bethanechol was suppressed by 31% in the presence of the Ca2+ -dependent Cl- -channel blocker A-9-C. The L-type voltage-operated Ca2+ channel blocker nifedipine decreased the maximal constrictor response to bethanechol by 59%, while the putative receptor-operated Ca 2+ channel blocker SK&F 96365 converted this vasoconstriction into vasodilation which was not affected by the nitric oxide synthase inhibitor L-NAME. Coronary vascular responses to bethanechol were almost abolished by a combination of nifedipine and SK&F 96365. The protein kinase C inhibitor chelerythruine reduced bethanechol-evoked peak vasoconstriction by 79%. The bethanechol-induced tonic vasoconstriction was rapidly converted into vasodilation by the concomitant infusion of SK&F 96365 or nifedipine, but the simultaneous infusion of chelerythrine gradually attenuated this response. These data suggest that the novel receptor-operated Ca2+ channel, voltage-operated Ca2+ channel, and protein kinase C are the most crucial signaling components for muscarinic receptor-mediated coronary vasoconstriction in the isolated rat heart. Extracellular Ca 2+ influx via receptor-operated Ca2+ channels and voltage-operated Ca2+ channels is essential to both phasic and tonic vasoconstrictor responses to bethanechol. Protein kinase C plays a pivotal role in the regulation of bethanechol-evoked vasoconstriction by sensitizing the contractile apparatus and modulating the activity of Ca 2+ channels.

The spinal cord is an important component in the processing and modulation of painful stimuli. Nerve signals from the periphery are relayed and further conducted to the brain (nociception) in the spinal cord, and the most essential modulation of painful information (antinociception) occurs here. Several neurotransmitters are involved in spinal pain modulation, among them acetylcholine. However, the role of acetylcholine has previously been little investigated. In the present thesis, the acetylcholine release in the spinal cord was studied in vivo. By using spinal microdialysis on anaesthetised rats, the effects on the intraspinal acetylcholine release of various receptor ligands and analgesic agents were examined. This, together with pain behavioural tests and in vitro pharmacological assays, was used to evaluate the role of acetylcholine in spinal pain modulation. The four studies in this thesis resulted in the following conclusions: An increased release of spinal acetylcholine is associated with an elevated pain threshold, while a decreased acetylcholine release is associated with hyperalgesia, as seen after systemic treatment with a muscarinic agonist and an antagonist. Lidocaine is a potent analgesic when given systemically. It was found to produce an increase of intraspinal acetylcholine after intravenous injection of analgesic doses. This effect was attenuated after muscarinic, and abolished after nicotinic, receptor blockade. Various a2-adrenergic ligands, associated with nociceptive or antinociceptive effects, were found to affect intraspinal acetylcholine release via action on nicotinic receptors. Finally, the involvement of spinal acetylcholine in the analgesic effects of aspirin and paracetamol was examined. It was found that spinal acetylcholine could participate in the analgesic effects of aspirin, but not of paracetamol. The present thesis provides data that clearly demonstrate a relationship between intraspinal acetylcholine and antinociception, and elucidate interactions between acetylcholine and other mechanisms that mediate antinociception in the spinal cord.

Tendinosis is a condition of chronic pain that afflicts several human tendons, not least the patellar tendon, in which case it is often clinically referred to as ‘jumper’s knee’. The exact mechanisms behind tendinosis are yet not fully understood. One draw-back in the case of patellar tendinosis has been the lack of knowledge of the innervation patterns of the human patellar tendon. It cannot be excluded that the processes of tendinosis are influenced by nerve mediators, released from nerve endings or from stimulated cells inside the tendon. Thus, the studies of the present thesis aimed to 1) map the general, sensory, cholinergic and sympathetic innervation patterns of the human patellar tendon, in both the tendon tissue proper and the loose paratendinous connective tissue surrounding the tendon, and 2) investigate the possible existence of a production of signal substances, traditionally associated with neurons, in non-neuronal tendon cells, and to see if there are signs of local cholinergic and catecholaminergic signaling pathways. Biopsies of both normal pain-free patellar tendons and patellar tendons from patients with chronic painful tendinosis were collected and investigated. The main method utilized was immunohistochemistry, using antibodies directed against synthesizing enzymes for acetylcholine and catecholamines, against muscarinic and adrenergic receptors, and against markers of general and sensory innervation. In situ hybridization (ISH) to detect mRNA for the cholinergic/catecholaminergic synthesizing enzymes was also used. It was found that the loose paratendinous connective tissue of the patellar tendon was rather richly innervated by nerve structures. These consisted of large nerve fascicles, as well as perivascular innervation in the walls of some of the larger arteries and smaller blood vessels. It was found that part of the nerve structures corresponded to sensory afferents, and that some conformed to cholinergic and, especially, sympathetic nerve fibers. The tendon tissue proper was strikingly less innervated than the paratendinous tissue. The sparse innervation that was found in the tendon tissue proper was seen in narrow zones of loose connective tissue and blood vessels, interspersed between the collagen bundles. The overall impression was that the patterns of distribution of the general, sensory, and autonomic innervations of tendinosis tendon tissue were similar to those of normal tendon tissue proper. The most pioneering findings were the immunohistochemical observations of an expression of enzymes related to production of both acetylcholine and catecholamines within the tendon cells (tenocytes) themselves, as well as of a presence of the receptors for these substances on the same cells; features that were predominantly seen in tendinosis tendons. The observations of the synthesizing enzymes for acetylcholine and catecholamines in tenocytes were confirmed by ISH findings of mRNA for these enzymes in the tenocytes. Immunoreactions for muscarinic and adrenergic receptors were also found in blood vessel walls and in some of the nerve fascicles. In summary, this thesis presents novel information on the innervation patterns of the human patellar tendon, in healthy individuals with pain-free tendons as well as in patients with chronic painful tendinosis. Furthermore, it gives the first evidence of the presence of a local, non-neuronal production in the tendon tissue of signal substances normally seen in neurons, and a basis for these substances to affect the tenocytes as these cells also display muscarinic and adrenergic receptors. Thus, the results indicate an existence of autocrine and/or paracrine cholinergic/catecholaminergic systems in the tendon tissue; systems that seem to be up-regulated in tendinosis. This is of great interest as it is known that stimulation of receptors for both catecholamines and acetylcholine can lead to cell proliferation, interfere with pain sensation, influence collagen production, and take part in vasoregulation, as well as, in the case of adrenergic receptors, promote cell degeneration and apotosis. All these processes represent biological functions/events that are reported to be affected in tendinosis. In conclusion, despite the fact that there is very limited innervation within the patellar tendon tissue proper, it is here shown that effects of signal substances traditionally associated with neurons seem to occur in the tissue, via a local production of these substances in tenocytes.

Neurotransmitter receptor types mediating the generation of intracellular signals are of two types; ligand-gated ion channels and G protein coupled receptors. The effector enzyme phosphoinositide-specific phospholipase C (PLC) is modulated by stimulation of G protein coupled receptors, leading to an increased breakdown of inositol phospholipids ("Ptdlns breakdown").In recent years, the receptors in the brain coupled to PLC and modulation of such receptor-mediated Ptdlns breakdown have been characterised. One such modulation is the "potassium effect", whereby an increase in the assay [K+] from 6 to 18 mM potentiates the Ptdlns breakdown response to the muscarinic agonist carbachol in the rat brain. It has been speculated that this effect is one way of enhancing the signal :noise ratio of muscarinic neurotransmission. The mechanisms responsible for the potassium effect have been studied in this thesis.Initial methodological studies indicated that the temperature of the Krebs buffer used after tissue dissection was an important factor regulating the Ptdlns response to receptor stimulation. Expressing the Ptdlns breakdown response as a fraction of the total labelled phosphoinositides was more useful than other ways of expressing the data. Acid extraction of the Lipid fraction was also superior to neutral extraction.Miniprismspreparedfrompig striatum and hippocampus showed qualitative (but not quantitative) similarities with the rat with respect to stimulation by carbachol, noradrenaline and the potassium effect. Dopamine also stimulated Ptdlns breakdown, though probably via a noradrenergic mechanism.The enhancing actions of potassium appeared to be selective for muscarinic Ml-type receptors. Thus glutamate, quisqualate and NaF-stimulated Ptdlns breakdown are not affected by raised [K+].The potassium effect is brought about by two mechanisms. In calcium-free Krebs buffer, the effect could be mimicked by the calcium channel agonist BAY K-8644 and partially antagonised by verapamil. At an assay [Ca2*] of 2.52 mM, however, modulation of calcium uptake had little effect on carbachol-stimulated Ptdlns breakdown at either normal or raised [K+]. The synergy between potassium and carbachol at252 mM Ca?+ is not dependent upon tissue depolarisation perse, since other ways of depolarising the tissue did not enhance the response to carbachol. It is suggested that potassium might have a direct effect on the muscarinic Ml-type receptor - G protein - PLC complex.In order to investigate this possibility, the effect of fluoride ions (which activate G proteins via formation of AlF4) on basal and carbachol-stimulated Ptdlns breakdown was investigated. Fluoride ions inhibited the enhanced breakdown response to carbachol found at raised [K+]. However, this effect is secondary to effects of fluoride on PLC substrate availibility rather than on G protein function. / digitalisering@umu

This work improved the radiosynthesis of a known M2 muscarinic receptor imaging agent, [18F]FP-TZTP, and subsequent syntheses and in vitro evaluation of several novel TZTP derivatives highlighted a lead compound which exhibited M4 potency and selectivity, the thioether fluoro-polyethyleneglycol, which was then adapted for radiolabelling (23% radiochemical yield (uncorrected), >99% radiochemical purity, reaction time of 37 minutes). The present study also seeked to utilize aziridines as intermediates in [18F]-radiolabelling chemistry for the facile radiosynthesis of [18F]-labelled beta-blockers. Novel [18F]-labelled amines were synthesized via ring-opening and deprotection to yield the [18F]-1-fluoro-2-propanamine moiety (85%) favourably over the regioisomer [18F]-2-fluoro-1-propanamine (15%). Subsequent attempts to use these amine synthons in the synthesis of the beta-blocker [18F]Exaprolol resulted in poor radiochemical yields (1-3%). The chemistry of aziridine ring-opening with 19F-fluoride sources was thoroughly explored in order to understand the fundamentals of this chemistry, and the 1-fluoro-2-propanamine moiety was characterized by X-ray crystallography and NMR spectroscopy.

This work improved the radiosynthesis of a known M2 muscarinic receptor imaging agent, [18F]FP-TZTP, and subsequent syntheses and in vitro evaluation of several novel TZTP derivatives highlighted a lead compound which exhibited M4 potency and selectivity, the thioether fluoro-polyethyleneglycol, which was then adapted for radiolabelling (23% radiochemical yield (uncorrected), >99% radiochemical purity, reaction time of 37 minutes). The present study also seeked to utilize aziridines as intermediates in [18F]-radiolabelling chemistry for the facile radiosynthesis of [18F]-labelled beta-blockers. Novel [18F]-labelled amines were synthesized via ring-opening and deprotection to yield the [18F]-1-fluoro-2-propanamine moiety (85%) favourably over the regioisomer [18F]-2-fluoro-1-propanamine (15%). Subsequent attempts to use these amine synthons in the synthesis of the beta-blocker [18F]Exaprolol resulted in poor radiochemical yields (1-3%). The chemistry of aziridine ring-opening with 19F-fluoride sources was thoroughly explored in order to understand the fundamentals of this chemistry, and the 1-fluoro-2-propanamine moiety was characterized by X-ray crystallography and NMR spectroscopy.