No description available.

The present thesis investigates the role of the prefrontal cortex and cholinergic modulation in attentional performance, and to a lesser extent, inhibitory response control, in rats. A greater understanding of these functions is important for the effective treatment of attentional and impulsive control deficits, present in a range of neuropsychiatric disorders. For this field to progress, the assessment of attentional performance in a similar manner across humans and animals is crucial. In the present thesis, attentional performance was assessed on the novel, touchscreen-based rodent continuous performance task (rCPT), which assesses sustained, focused attention in essentially an identical manner to CPTs commonly used in the clinic. Findings were compared to performance on the well-characterised 5-choice serial reaction time task (5-CSRTT), which assesses sustained, spatial divided attention and shares some, but not all characteristics of CPTs. The series of experiments described in this thesis contributes to the understanding of the role of the prefrontal cortex and cholinergic modulation in attentional performance; they also highlight differences between the two tasks in behaviour, brain functions and networks. Excitotoxic lesions of the medial prefrontal cortex (mPFC) and a range of cholinergic systemic pharmacology validated the role of the prefrontal cortex and cholinergic modulation in rCPT performance. A chemogenetic study also validated the role of the ascending cholinergic basal forebrain system in 5-CSRTT performance. These findings support 1. the idea of the relationship between cholinergic system activation and attentional performance to resemble an ‘inverted-U’ shaped function; 2. a double dissociation of mPFC sub-regions on attentional performance, in which the prelimbic cortex (PL) appears to play a role in rCPT performance, compared with a role of the anterior cingulate cortex (ACC) in 5-CSRTT performance; and 3. a role of ascending cholinergic projections from the basal forebrain to the ACC in 5-CSRTT performance. These findings also establish the development of a successful flanker distractor probe in rodents on the rCPT. This thesis concludes with an important comparison of the attentional and impulsivity measures in the rCPT compared to the 5-CSRTT, to help provide guidelines as to which task is most appropriate to use for particular research questions.

Adolescence is a transitional period between childhood and adulthood, in which the individual undergoes significant cognitive, behavioral, physical, emotional, and social developmental changes. During this period, adolescents engage in experimentation and risky behaviors such as licit and illicit drug use. Adolescents' high vulnerability to abuse drugs and natural reinforcers leads to greater risk for developing substance use disorders (SUDs) during adulthood. Accumulating evidence indicates that the use and abuse of licit and illicit drugs during adolescence and emerging adulthood can disrupt the cholinergic system and its processes. This review will focus on the effects of peri-adolescent nicotine and/or alcohol use, or exposure, on the cholinergic system during adulthood from preclinical and clinical studies. This review further explores potential cholinergic agents and pharmacological manipulations to counteract peri-adolescent nicotine and/or alcohol abuse.

No description available.

Repeated stress has been reported to cause reversible impairment to the hippocampus. Glutamatergic and cholinergic systems were proposed to be involved in responses seen after exposure to stress and cholinesterase inhibitors. Effects of concurrent exposure to repeated stress and chlorpyrifos (CPF) on concentrations of excitatory amino acids, activities of cholinergic enzymes, and maximum binding density (Bmax) and equilibrium dissociation rate constant (Kd) of NMDA and total muscarinic receptors were studied in Long-Evans rats. The study was divided into 4 experiments. The first experiment was to find the dose of CPF to use for studies on the interaction of stress and CPF. From the results obtained, 60% of the maximum tolerated dose was chosen. An experiment to determine effects of repeated stress and CPF on cholinergic enzymes and glutamate included groups of rats (n=7-8) that were handled 5 days/week; restrained 1 hour/day for 5 days/week; swum 30 minutes for 1 day/week; or restrained 4 days/week and swum for 1 day/week, for 28 days. On day 24, each group was injected either with corn oil or CPF 160 mg/kg sc 4 hours after restraint. On day 28, blood samples were collected for acetylcholinesterase (AChE) activity. Brains were dissected into hippocampus (HP) and cerebral cortex (CC) to determine activities of acetylcholinesterase (AChE), carboxylesterase (Cbxy), and choline acetyltransferase (ChAT), and glutamate and aspartate concentrations. CPF inhibited AChE activity in blood, CC and HP, but stress did not affect AChE activity. Repeated restraint with swim reduced Cbxy and CPF inhibited Cbxy. Restraint with swim had a statistical trend to increase concentrations of glutamate in the HP more than swim alone (p = 0.064); but CPF had no effect on glutamate in the HP. CPF decreased concentrations of elevated aspartate in the HP of rats that were restrained and swum. The results suggested that restraint with swim indirectly elevated acetylcholine in the CC, and tended to increase glutamate in the HP. The experiment designed to study the effects of concurrent exposure to stress and CPF on NMDA and total muscarinic receptors was designed similar to the previous study, except that endpoints were Bmax and Kd of NMDA and total muscarinic receptors in the HP and CC, and NMDA receptors in the hypothalamus (HT). Restrained rats had higher Kd of NMDA receptors in the HP than control and restrained with swim rats; however, Bmax was similar. CPF deceased Bmax and Kd of total muscarinic receptors in the CC of swum rats (237.64 &#177; 17.36 fmol/mg protein, 0.216 &#177; 0.023 nM) and CPF also decreased Bmax of total muscarinic receptors in the CC of restrained rats (229.08 &#177; 17.36 fmol/mg protein). There were no effects of stress, CPF, or interactions of stress and CPF on NMDA receptors in the CC or on total muscarinic receptors in the HP. In summary, CPF was capable of modulation of total muscarinic receptors of swum and restrained rats, suggesting that cholinergic transmission in the CC for cognition, sensory and motor activity may be modified. Furthermore, we examined effects of stress and CPF on concentrations of monoamines. Swim stress and CPF individually decreased concentrations of norepinephrine in the HP, whereas swim and restraint with swim decreased concentrations of norepinephrine but increased concentrations of DOPAC in the HT. Swim stress increased concentrations of dopamine in the HT more than control or restraint. CPF did not alter concentrations of norepinephrine, dopamine, or DOPAC in the HT. The interactions of repeated stress and CPF on serotonin approached significance in the HP (p = 0.06) and HT (p = 0.08). CPF increased serotonin concentrations in rats that were handled and restrained but not swum. CPF reduced the elevated concentrations of serotonin in restrained rats and restrained with swim rats (p < 0.05). Swim and restraint with swim were potential stress models that altered noradrenergic, dopaminergic, and serotonergic responses in the HT. In summary, repeated stressors had effects on glutamatergic, cholinergic, and monoamine systems. CPF had effects on cholinergic and monoamine systems but the interactions between stress and CPF were few. / Ph. D.

Kateřina Janská Evaluation of the kinetics of acetylcholinesterase inhibitors in vitro Diploma thesis Charles University in Prag, Faculty of Pharmacy in Hradec Králové Pharmacy Department of Biological and Medical Sciences Supervisor: Doc. MUDr. Josef Herink, DrSc. Consultant: PharmDr. Vendula Šepsová The aim of the thesis was to determine the type of an inhibition of newly synthesized AChEI and to find out if AChEI structure changes influence the type of an inhibition. Altogether 12 substances (7 tacrine hybrides and 5 7-methoxy- donepezil hybrids) were investigated. The inhibition potential of the tested substances was studied in vitro on the human recombinant AChE. Spectrophotometric Ellman method was utilized as the measurement tool. The noncompetitive type of an inhibition for substances EN 1-5, PC-25 and PC-33, mixed type of an inhibition for substances PC-48 and PC-49, uncompetitive type of an inhibition for substances EN-6, EN-7 and competitive type of an inhibition for the substance PC-37 was determined. The greatest inhibition potential according to Ki values were found for substances EN-7 and PC-37. Substances PC-37 and PC-48 were determined as substances with the biggest affinity to the AChE. The type of an inhibition has been influenced by a substituent position in PC substances and by...

Adéla Dolejšová Testing the efficacy of potential therapeutics for Alzheimer,s disease Charles University in Prague, Faculty of Pharmacy in Hradec Kralove Pharmacy The aim of this thesis was to find out whether already implemented Ellman's method, which is used to analyse irreversible inhibitors AChE, is going to be a suitable technique for measuring reversible inhibitors. Furthermore, the efficiency of newly synthesized AChE inhibitors was established. These inhibitors will be used for treating AD or as prophylaxis against neural paralytic substances. Moreover, it was compared affect the efficacy of AChE inhibitors after intramuscular and intraperitoneal administration. The measurements were done in vivo on potkan species Wistar. In the first experiment standard AChE (tacrin, 7-MEOTA, donepezil, rivastigmin) inhibitors were applied to verify the method. In the second experiment the newly synthesizes AChE (K 298, K 344, K 474) inhibitors were medicine. The results confirmed the effectiveness of the tested method on commonly used inhibitors such as donepezil and rivastigmin. Out of the newly synthesized K 298, K 344 and K 474 inhibitors none was proven to have any significant inhibiting activity. Key words: acetylcholinesterase, acetylcholinesterase inhibitors, Alzheimer disease, cholinergic system,...

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.

O sistema colinérgico está sabidamente envolvido com processos cognitivos. Em trabalho recente mostramos que a infusão do peptídeo A promoveu neurodegeneração e redução da memória de ratos. O treino semanal dos animais em equipamento de esquiva ativa recuperou o desempenho na tarefa e aumentou a densidade de receptores nicotínicos &alpha;7 em áreas relacionadas à memória. No presente trabalho, o antagonismo de &alpha;7 com metilicaconitina (MLA), em camundongos, promoveu perda cognitiva, porém a recuperação com o treino foi parcial. A infusão conjunta de &beta;A e MLA causou perda da memória, mas essa não foi revertida com o treino semanal. Os animais com MLA apresentaram aumento da atividade da acetilcolinesterase (AChE) e aumento de BDNF, que poderia ser relacionado à resiliência a injúrias. Porém, animais com A e MLA apresentaram aumento da atividade da AChE e redução de BDNF, sugerindo perda dos mecanismos de neuroproteção deflagrados por &alpha;7. Com isso, sugere-se que &alpha;7 tenha um papel determinante na recuperação da memória e resiliência tecidual, frente à neurodegeneração. / Cholinergic system plays an important role in cognitive processes. In a recent work we showed that infusion of A&beta; promoted neurodegeneration and reduction of memory of rats. Week training of animals in active avoidance shuttle box recovered their performance and increased the density of &alpha;7 nicotinic receptors in brain areas related to memory. In the present work, infusion of the &alpha;7 antagonist methyllycaconitine (MLA), in mice, caused cognitive impairment, but memory recover with week training was partial. Infusion of &beta;A together with MLA promoted memory loss, but this was not recovered with the week training. MLA infused mice presented increase in acetylcholinesterase (AChE) activity and increase in BDNF, which could be related to resilience to tissue injuries. However, animals infused with &beta;A and MLA showed increase in AChE activity and reduction of BDNF, suggesting loss of neuroprotection mechanisms triggered by &alpha;7. It is suggested that &alpha;7 has a determinant role in memory recover and brain resilience, in neurodegenerative processes.

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.