Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dementia is a brain disorder characterized by decline in several mental functions and results in deficits in memory functioning in a variety of cognitive tasks. Evidence has emerged showing that anthocyanins (ANT) possess antioxidant, vasodilator and neuroprotective properties, and more recently are able to improve memory. The aim of our study was to investigate the role of ANT on memory and relate these changes to the cholinergic and purinergic in a model of amnesia induced by scopolamine (SCO), in a model of sporadic dementia of the Alzheimer type (SDAT), induced by intracerebroventricular injection of streptozotocin (ICV-STZ). We used male adult rats, which were previously treated with an extract rich in ANT (200mg/kg) for seven days. For each study protocol, we used the same dose of ANT only varying the time of drug administration SCO ip (1mg/kg) and ICV-STZ (3mg/kg) specific to the models of amnesia and dementia, respectively. The results showed that pretreatment with ANT reversed the memory deficits in an amnesia model and also in the model of SDAT. Regarding the activity of AChE enzyme was observed a significant increase in SCO group accompanied by a decrease in the activity of Na+, K+-ATPase and Ca2+-ATPase, and these effects were prevented by ANT treatment. It was also observed an increase in NTPDase activity, decreased in ADA and 5'-NT activity from brain of animals injected with SCO, accompanied by a reduction in the ATP and adenosine levels, which can compromise the purinergic signaling. ANT treatment was able to prevent changes in NTPDase, ADA and 5 -NT. The ICV-STZ treated animals showed a high anxiogenic-like behavior and ANT treatment prevented this effect, indicating that it can be seen with anxiolytic properties molecules, as in vitro experiments demonstrated their affinity for the benzodiazepine site of GABAA receptors. The animals ICV-STZ treated showed marked increase in AChE activity and Ca2+-ATPase, and decreased activity of Na+, K+-ATPase, causing disturbances in electrolyte concentrations of Na+ and Ca2+ and this could lead to neuronal excitotoxic and increased activity AChE in SDAT, and these effects were prevented by administration of ANT. Moreover, both the density and immunoreactivity for 5'-NT was markedly reduced in animals ICV-STZ as compared with the group of -normal rats. In addition, ICV-STZ treatment showed a significant decrease in 5'-NT and NTPDase activity in a model of SDAT. In the hippocampus of ICV-STZ animals was found considerable increase in levels of ROS total, MDA and NOx, indicating marked oxidative stress, which could lead to a decrease in neuronal viability and NTPDase and 5'-NT activity, since they are linked to the cell membrane. In this context, we suggest that both cholinergic and purinergic systems can be regulated by ANT treatment, thus defining use of this compound as a new strategy to control the deterioration of memory in aging associated with these systems. / A demência é uma desordem cerebral caracterizada por um declínio em várias funções mentais e resulta em déficits no funcionamento da memória e em uma variedade de tarefas cognitivas. Evidências têm surgerido que as antocianinas (ANT) possuem propreidades antioxidantes, vasodilatadoras e neuroprotetoras, sendo capazes de melhorar a memória. O objetivo do nosso trabalho foi investigar o papel das ANT sobre a memória e, relacionar estas mudanças com o sistema colínergico e purinérgico em um modelo de amnésia induzido por escopolamina (SCO), em um modelo de demência esporádica do tipo Alzheimer (DAS), induzido por administração intracerebroventricular de streptozotocina (ICV-STZ). Foram utilizados ratos machos adultos, os quais foram tratados previamente com um extrado rico em ANT (200mg/kg) durante sete dias. Para cada protocolo de estudo, foi utilizada a mesma dose de ANT variando apenas o tempo de administração das drogas SCO ip (1mg/kg) e ICV-STZ (3mg/kg) especificas para aos modelos de amnésia e de demência, respectivamente. Os resultados obtidos demonstraram que o tratamento prévio de ANT reverteu déficits de memória no modelo de amnésia e no modelo de DAS. Em relação à atividade da enzima AChE foi observado um aumento significativo nos grupos SCO acompanhado por uma diminuição da atividade das enzimas Na+,K+-ATPase e Ca2+-ATPase, e estes efeitos foram prevenidos pelo tratamento com ANT. Foi também observado aumento na atividade da NTPDase, diminuição na atividade da ADA e 5 -NT no cérebro de animais injetados com SCO, acompanhado por uma redução nos niveis de ATP e adenosina, ao qual pode comprometer a sinalização purinérgica. E o tratamento com ANT impediu alterações na atividade destas enzimas. Os animais ICV-STZ tratados apresentaram elevado comportamento ansiogênico e o tratamento com ANT previniu este efeito, indicando que as ANT podem ser consideradas moléculas com propriedades ansiolíticas, uma vez que experimentos in vitro, mostraram sua afinidade pelo sítio benzodiazepínico de receptores GABAA. Os animais tratados com ICV-STZ apresentaram marcante elevação na atividade da AChE e Ca2+-ATPase, e redução na atividade da Na+,K+-ATPase, causando perturbações nas concentrações eletrolíticas de Na+ e de Ca2+ e isto poderia levar a excitotoxicidade neuronal e aumento na atividade da AChE na DAS, e estes efeitos foram prevenidos pela administração de ANT. Além disso, tanto a densidade como a immunorreatividade para a 5 -NT foi marcadamente reduzida em animais ICV-STZ em comparação com o grupo de ratos normais. Além disso, o tratamento com STZ mostrou uma diminuição significativa para a atividade da NTPDase e 5 -NT no modelo em animais ICV-STZ tratados. No hipocampo de animais ICV-STZ tratados foi encontrado uma elevação considerável nos níveis de ROS total, MDA e NOx, indicando marcante estresse oxidativo, que poderia levar a diminuição na viabilidade de neurônios e na atividade das enzimas NTPDase e 5 -NT, uma vez que elas estão ligadas a membrana celular. Neste contexto, podemos sugerir que tanto o sistema colinérgico como purinérgico podem ser regulados pelo tratamento com antocianinas, definindo assim, o uso deste composto como uma nova estratégia para controlar a deteriorização mnemônica no envelhecimento associado a esses dos sistemas.

Dès les stades précoces du développement et jusqu’à l’âge adulte, le facteur de transcription TSHZ3 est fortement exprimé dans les neurones pyramidaux (PNs) du cortex. Les PNs de la couche V forment la synapse cortico-striée en contactant les neurones épineux moyens (MSNs) du striatum. A ce niveau, l’expression de TSHZ3 n’est pas retrouvée dans les MSNs mais dans les interneurones cholinergiques (CINs). Des données récentes ont établi un lien entre délétion hétérozygote du gène TSHZ3/Tshz3, troubles du spectre autistique (TSA) et dysfonctionnement de la circuiterie cortico-striée (Caubit et al., Nat Genet 2016). Afin de mieux comprendre le rôle de TSHZ3 dans la circuiterie cortico-striée, nous avons caractérisé deux modèles murins de délétion conditionnelle de Tshz3, ciblant soit les neurones de projection à partir de la période postnatale (souris Tshz3-pnCxKO), soit les neurones cholinergiques à partir de la période embryonnaire (souris Tshz3-ChATCre). Chez les souris Tshz3-pnCxKO, la perte de TSHZ3 entraîne une moindre excitabilité des PNs de la couche V, ainsi qu’une diminution de la probabilité de libération du glutamate par leurs afférences. Nous montrons également une profonde altération du fonctionnement de la synapse cortico-striée. Chez les souris Tshz3-ChATCre, nous montrons que la perte de Tshz3 modifie les propriétés membranaires et de décharge d’une proportion des CINs, qui sont les seuls neurones cholinergiques de l'encéphale exprimant TSHZ3 de façon importante. Ces changements fonctionnels suggèrent que TSHZ3 joue un rôle clé dans le développement des PNs du cortex, de la voie cortico-striée et des CINs, confirmant son implication dans les TSA. / The zinc-finger transcription factor TSHZ3 is highly expressed by cortical projection neurons (PNs) from embryonic stages to adulthood, including layer V pyramidal neurons that project to the striatum. There, TSHZ3 is expressed by cholinergic interneurons (CINs) but not by the main targets of PNs, i.e. the medium spiny neurons. Interestingly, recent evidences link heterozygous TSHZ3/Tshz3 gene deletion to autism spectrum disorder (ASD) and to corticostrial circuitry dysfunction (Caubit et al., Nat Genet 2016). In order to provide further insights on the role of Tshz3 in the corticostriatal circuitry, we have characterized two conditional KO mouse models in which its expression is lost either in projection neurons at early postnatal stage (Tshz3-pnCxKO) or in cholinergic cells beginning at embryonic stage (Tshz3-ChATCre). In Tshz3-pnCxKO mice, we confirmed that Tshz3 expression is lost in glutamatergic PNs without altering their number. Our electrophysiological study revealed that layer V PNs are less excitable and that glutamate release probability from their afferents is decreased. We also found dramatic changes of both corticostriatal synaptic transmission and plasticity. In ChAT-Cre mice, we found that Tshz3 is expressed in the striatum by almost 100% of CINs, while it is little or no expressed in the other cholinergic nuclei of the brain. Interestingly, the loss of Tshz3 impacts the spontaneous firing pattern of a subpopulation of CINs without altering their number. These functional changes suggest that TSHZ3 plays a key role in PNs, corticostriatal pathway and CINs development, supporting its implication in ASD.

La maladie d’Alzheimer est souvent précédée de troubles cognitifs légers (MCI) associés à la lésion du cortex entorhinal, une région interconnecté avec l’hippocampe. Cependant, un tiers des patients MCI présentent une rémission cognitive suggérant l’action de mécanismes compensatoires. Ces mécanismes pourraient être déficitaires chez les patients porteurs de l’allèle APOE4 présentant un MCI plus agressif.Par des approches multidisciplinaires chez la Souris, ce travail a démontré que la lésion entorhinale induit des déficits comportementaux et une hyperactivité de l’hippocampe. Or, le bourgeonnement des fibres cholinergique dans l’hippocampe compense ces déficits. Comme le bourgeonnement cholinergique est inhibé en présence de l’APOE4, cela pourrait contribuer au déclin cognitif exacerbé de ces patients. Par conséquent, l’inhibition de l’hyperactivité hippocampique chez les 50% de patients APOE4 représente une alternative prometteuse pour le traitement symptomatique du MCI. / Mild Cognitive Impairments (MCI) often precedes Alzheimer’s disease (AD) and is characterized by the loss of entorhinal neurons leading to a hippocampal disconnection. However, MCI patients also revert to normal cognition, suggesting compensatory mechanisms that alter the disease progression. This compensation may be impaired in patients bearing the APOE4 allele that are more prone to MCI, present less cognitive reversion and faster transition to AD.This work in mice, demonstrated that the sprouting of cholinergic fibers compensates entorhinal lesions through the reduction of the related hippocampal hyperactivity. As in APOE4 mice the cholinergic sprouting was altered in association with cognitive impairments, such impaired synaptic compensation may contribute to the faster cognitive decline of these patients. Therefore, supporting or mimicking the cholinergic control on hippocampal hyperactivity may represent a promising alternative therapeutic strategy for APOE4-carriers.

The development of cholinergic neurons in mouse frontal cortex was studied both in vivo and in vitro by immunocytochemistry with an antibody to choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis. While cortical cholinergic neurons have previously been characterized in rat cortex, up until very recently, intrinsic cortical cholinergic neurons were considered to be absent in mouse, and little is known about their development or phenotypic characteristics. The present study found no ChAT-positive neurons in mouse frontal cortex on postnatal day 0 (P0, the day of birth). On P7 there were few, faintly stained, ChAT-positive neurons. The numerical density of ChAT-positive neurons increased substantially with age, from none on P0, to 9.2 + 1.4 on P7, to 14.8 + 0.9 on P16, and 41.6 + 3.9 in adulthood. Considering that the numerical density of total neurons decreases during this postnatal period, the data represent a marked developmental increase in the percentage of cholinergic neurons. The development of cholinergic neurons showed very similar timelines in rat and mouse frontal cortex. Cultures prepared from mouse frontal cortex on embryonic day 16 were maintained for 25, 76, or 100 days in vitro (div). The percentage of ChAT-positive neurons was considerably higher than in vivo, ranging from a mean 28% to 31% across the three age (div) groups. With increasing age of the cultures, the numerical density of total neurons and ChAT-positive neurons decreased while the percentage of ChAT-positive neurons did not change significantly. These observations suggest some temporal stability in the cultures. Using dual immunofluorescence, ChAT-positive neurons were tested for colocalization with GAD or TH. The majority of ChAT-positive neurons colocalized with GAD, both in vitro and in vivo. However, ChAT did not colocalize with TH, either in vitro or in vivo. Our comparison of intact frontal cortex and cultures suggest that while the percentage of cholinergic neurons was greater in the cultures, the cholinergic neurons developed phenotypic similarities in vitro and in vivo.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23340号 / 生博第458号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Environs 70 millions de personnes seront atteintes par la maladie d’Alzheimer (MA) en 2030 et les coûts engendrés par la prise en charge des patients affectés par cette pathologie excédera quatre fois les coûts des maladies cancéreuses. Dans le but lutter contre cette pathologie multifactorielle, des stratégies thérapeutiques combinatoires ainsi que le développement de MTDL « Multi Target Directed Ligands » suscitent un grand intérêt. Dans ce contexte, l’association d’inhibiteurs de l’acétylcholinestérase (AChE) avec les agonistes des récepteurs sérotoninergiques de type 4 (5-HT4) a montré des effets bénéfiques sur la mémoire et la protection du cerveau chez les rongeurs. Après avoir démontré l’avantage de l’association de la galantamine, un inhibiteur de l’AChE avec le RS 67333, un agoniste des récepteurs 5-HT4 dans un modèle de déficit mnésique induit par la scopolamine sur les performances de mémoires de travail et de référence, nous avons étudié le donecopride, un MTDL récemment développé, à la fois inhibiteur de l’AChE et agoniste partiel des récepteurs 5-HT4. Chez les souris sauvages, une administration unique de donecopride augmente la sécrétion du neuropeptide protecteur sAPPα et a démontré des effets anti-amnésiants et pro-cognitifs. Dans un modèle amyloïdogénique de la MA (5xFAD), six mois de traitement chronique par le donecopride ont amélioré les performances de mémoire de travail à l’âge de 9 mois, sans modifier la densité des plaques amyloïdes au niveau cérébral. Par ailleurs, nous avons également étudié les effets d’un autre composé MTDL « 7m » qui est à la fois agoniste des récepteurs 5-HT4 et antagoniste des récepteurs 5-HT6, qui a montré des effets anti-amnésiants chez les souris sauvages lorsque le déficit mnésique est induit par administration de scopolamine. Ainsi, la modulation chronique et simultanée de différentes cibles centrales d’intérêt représente un potentiel thérapeutique important dans le cadre du traitement de la MA. / In 2030, the number of people affected by Alzheimer’s disease (AD) is expected to reach almost 70 million, and the cost of the disease would be over four folds cost of cancer diseases care. To face the multifactorial disease combining drugs arouse a big interest and the concept of MTDL “Multi Target Directed Ligands” has emerged. Thus, combining acetylcholinesterase (AChE) inhibitors with serotonergic receptor type 4 (5-HT4) showed beneficial effects on memory and brain protection in rodents. First, we showed beneficial effects of galatamine, an AChE inhibitor while combined to RS 67333, a 5-HT4 receptors partial agonist, on working and reference memory performances in a pharmacological model with scopolamine-induced amnesia. Then, we assessed effects of donecopride, the AChE inhibitor and partial agonist of 5-HT4 receptors. Donecopride, which increases sAPPα release in cell culture and C57BL/6 mice, showed anti-amnesiant and pro-cognitive effects in NMRI mice. In 5xFAD mice, an amyloidogenic mice model of AD, six months of chronic treatment of donecopride improves working memory performances in 9 months old 5xFAD mice, but does not affect spatial learning performances neither flexibility. Moreover, donecopride inhibits AChE and does not affect 5-HT4 receptor expression level neither amyloid plaque density in the brain. In the other hand, we also showed that the MTDL, which modulate 5-HT4 and 5-HT6 receptors, recovers working memory performances in NMRI mice where scopolamine induced memory deficit. Thus, hitting multiple targets, particularly while administered early and chronically, accounts for a hopeful strategy to better face AD

The spleen is an important site for central regulation of immune function by noradrenergic sympathetic nerves, but little is known about this major region of neuroimmune communication in humans. Experimental studies using animal models have established that sympathetic innervation of the spleen is essential for cholinergic anti-inflammatory responses evoked by vagal nerve stimulation, and clinical studies are evaluating this approach for treating inflammatory diseases. Most data on sympathetic nerves in spleen derive from rodent studies, and this work has established that remodeling of sympathetic innervation can occur during inflammation. However, little is known about the effects of sepsis on spleen innervation. Our primary goals were to (i) localize noradrenergic nerves in human spleen by immunohistochemistry for tyrosine hydroxylase (TH), a specific noradrenergic marker, (ii) determine if nerves occur in close apposition to leukocytes, and (iii) determine if splenic sympathetic innervation is altered in patients who died from end stage sepsis. Staining for vesicular acetylcholine transporter (VAChT) was done to screen for cholinergic nerves. Archived paraffin tissue blocks were used. Control samples were obtained from trauma patients or patients who died after hemorrhagic stroke. TH + nerves were associated with arteries and arterioles in all control spleens, occurring in bundles or as nerve fibers. Individual TH + nerve fibers entered the perivascular region where some appeared in close apposition to leukocytes. In marked contrast, spleens from half of the septic patients lacked TH + nerves fibers and the average abundance of TH + nerves for the septic group was only 16% of that for the control group (control: 0.272 ± 0.060% area, n = 6; sepsis: 0.043 ± 0.026% area, n = 8; P < 0.005). All spleens lacked cholinergic innervation. Our results provide definitive evidence for the distribution of noradrenergic nerves in normal human spleen and the first evidence for direct sympathetic innervation of leukocytes in human spleen. We also provide the first evidence for marked loss of noradrenergic nerves in patients who died from sepsis. Such nerve loss could impair neuroimmunomodulation and may not be limited to the spleen.

The neurotrophic factor neurturin is required for normal cholinergic innervation of adult mouse heart and bradycardic responses to vagal stimulation. Our goals were to determine effects of neurturin deletion on development of cardiac chronotropic and dromotropic functions, vagal baroreflex response, and cholinergic nerve density in nodal regions of postnatal mice. Experiments were performed on postnatal C57BL/6 wild-type (WT) and neurturin knockout (KO) mice. Serial electrocardiograms were recorded noninvasively from conscious pups using an ECGenie apparatus. Mice were treated with atenolol to evaluate and block sympathetic effects on heart rate (HR) and phenylephrine (PE) to stimulate the baroreflex. Immunohistochemistry was used to label cholinergic nerves in paraffin sections. WT and KO mice showed similar age-dependent increases in HR and decreases in PR interval between postnatal days (P) 2.5 and 21. Treatment with atenolol reduced HR significantly in WT and KO pups at P7.5. PE caused a reflex bradycardia that was significantly smaller in KO pups. Cholinergic nerve density was significantly less in nodal regions of P7.5 KO mice. We conclude that cholinergic nerves have minimal influence on developmental changes in HR and PR, QRS, and QTc intervals in mouse pups. However, cholinergic nerves mediate reflex bradycardia by 1 week postnatally. Deletion of neurturin impairs cholinergic innervation of the heart and the vagal efferent component of the baroreflex early during postnatal development.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc) underlies the rewarding properties of ethanol (EtOH) and nicotine (NIC). Although the dogma is that EtOH enhancement of DA neural activity contributes to enhancement of DA transmission, DA neurons are not sensitive to rewarding levels of EtOH. However, VTA GABA neurons are sensitive to low-dose EtOH. We have shown previously that EtOH modulation of DA release in the NAc is mediated by α6-containing nicotinic receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and α6*-nAChRs may be a molecular target for low-dose EtOH. Thus, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on VTA GABAergic input to CINs and DA release in the NAc. Using DIO channel rhodopsin-2 (ChR2) viral injections into the VTA of VGAT Cre mice, we found that VTA GABA neurons send an inhibitory projection to CINs, replicating what has been demonstrated by others. This study investigated the acute and chronic effects of EtOH at this synapse. We demonstrate that EtOH markedly enhances CIN firing rate and that these effects are blocked by the α6-conotoxin MII (α-Ctx MII), knockout of accumbal α6*-nAChRs with α6-shRNA, and atypical GABA receptor antagonists. This study also investigated plasticity at this synapse. We demonstrate that a low frequency stimulation (LFS; 1 Hz, 240 pulses) causes inhibitory long-term depression at this synapse (CIN-iLTD) which is also blocked by α-Ctx MII, α6-shRNA, and atypical GABA receptor antagonists. We also show that CIN-iLTD is blocked in EtOH-dependent mice. Taken together, these findings suggest that EtOH affects the VTA GABAergic projection to CINs via α6*-nAChRs and that atypical GABA receptors also play a role.

Substance P (SP) evokes bradycardia that is mediated by cholinergic neurons in experiments with isolated guinea pig hearts. This project investigates the negative chronotropic action of SP in vivo. Guinea pigs were anesthetized with urethane, vagotomized and artificially respired. Using this model, IV injection of SP (32 nmol/kg/50 μl saline) caused a brief decrease in heart rate (-30 ± 3 beats/min from a baseline of 256 ± 4 beats/min, n = 27) and a long-lasting decrease in blood pressure (-28 ± 2 mmHg from baseline of 51 ± 5 mmHg, n = 27). The negative chronotropic response to SP was attenuated by muscarinic receptor blockade with atropine (-29 ± 9 beats/min before vs -8 ± 2 beats/min after treatment, P = 0.0204, n = 5) and augmented by inhibition of cholinesterases with physostigmine (-23 ± 6 beats/min before versus -74 ± 20 beats/min after treatment, P = 0.0250, n = 5). Ganglion blockade with chlorisondamine did not diminish the negative chronotropic response to SP. In another series of experiments, animals were anesthetized with sodium pentobarbital or urethane and studied with or without vagotomy. Neither anesthetic nor vagotomy had a significant effect on the negative chronotropic response to SP (F3,24 = 1.97, P = 0.2198). Comparison of responses to 640 nmol/kg nitroprusside and 32 nmol/kg SP demonstrated that the bradycardic effect of SP occurs independent of vasodilation. These results suggest that SP can evoke bradycardia in vivo through stimulation of postganglionic cholinergic neurons. Copyright (C) 1999 Elsevier Science Inc.