Envolvimento do sistema purinérgico no modelo de isolamento social em ratos Wistar adultos

O período do neurodesenvolvimento é caracterizado pela ampla estruturação do sistema nervoso central (SNC) e, portanto, é um momento de grande vulnerabilidade a agentes estressores endógenos e exógenos. Estresses ambientais como infecções durante a gravidez, adversidades na infância e isolamento do convívio social podem prejudicar a maturação do SNC, podendo, em última instância, servir como gatilho para o desenvolvimento de transtornos psiquiátricos. Roedores submetidos ao modelo de isolamento social pós-desmame apresentam prejuízos cognitivos, no filtro sensório-motor, na neurotransmissão de dopamina e de plasticidade sináptica. O sistema purinérgico é mediado pela sinalização extracelular de nucleotídeos e nucleosídeos. Com relação aos nucleotídeos de adenina, o ATP é hidrolisado até adenosina por ação de uma eficiente cadeia enzimática chamada de ectonucleotidases. Os nucleotídeos e nucleosídeos podem atuar nos receptores purinérgicos, que são divididos em receptores P1 e P2. Os receptores P2 são ainda subdivididos em P2X e P2Y. No SNC, as purinas exercem inúmeras funções fisiológicas e patológicas. Há uma evidente sobreposição dos mecanismos disfuncionais apresentados por animais socialmente isolados e pelas modulações que o sistema purinérgico exerce. Levando em consideração tais evidências, a presente dissertação teve como objetivo principal verificar se o isolamento social induz alterações na sinalização purinérgica em diferentes estruturas cerebrais de ratos. Para isso, ratos Wistar de 21 dias foram isolados socialmente por 8 semanas, e em seguida, verificamos diferentes fenótipos comportamentais e parâmetros neuroquímicos relacionados com o sistema purinérgico foram conduzidos. Primeiramente, foi observado prejuízos no filtro sensório-motor e aumento da interação social com ratos desconhecidos nos ratos isolados socialmente. Quanto os parâmetros neuroquímicos, nossos resultados mostraram um acúmulo de ADP no líquido cefalorraquidiano e um aumento da hidrólise do ADP em sinaptossomas de hipocampo e estriado nos animais que foram criados isoladamente. Por último, nas análises de expressão gênica, observamos diferentes alterações em diferentes estruturas cerebrais. O córtex pré-frontal apresentou redução na expressão gênica de adora2a, p2ry1 e p2rx5 nos animais que passaram pelo isolamento social. O hipocampo, por sua vez, apresentou regulação positiva dos genes p2ry4, p2ry13 e p2ry14 nos mesmos animais. Foi observado, ainda, que o estriado foi mais afetado pelo isolamento social, uma vez os animais submetidos ao isolamento apresentaram uma modulação negativa dos genes adora2a, p2rx4, p2ry2, p2ry6, p2ry12, p2ry13, entpd1, entpd2 e entpd3. Desta forma, nossos resultados demonstraram que o isolamento social pós-desmame promove abundantes alterações na sinalização purinérgica. Baseado em dados existentes na literatura, os dados obtidos nesse estudo podem indicar, no córtex pré-frontal, alterações de receptores envolvidos em processos cognitivos e de neurotransmissão de dopamina e glutamato. No hipocampo, apesar da escassez de estudos acerca desses receptores, parece haver indícios de disfunção microglial. O estriado, por sua vez, apresentou alterações de receptores associados, principalmente, com modulação de dopamina e disfunção microglial.The neurodevelopmental period it is characterized by wide structuring of central nervous system (CNS), which implies in highly susceptibility to endogenous or exogenous stressors. Environmental stressor in early-life, such as maternal separation, childhood adversities or social isolation, can disrupt brain development prompting psychiatric diseases. Post-weaning social isolation in rodents causes impairments in cognition, sensorimotor gating, dopamine neurotransmission and synaptic plasticity. The purinergic system is mediated by extracellular signaling of nucleotides and nucleosides. The ATP is hydrolyzed to adenosine by an efficient enzyme chain named as ectonucleotidases. The nucleotides and nucleosides can act on the purinergic receptors, which are divided in P1 and P2 receptors. P2 receptors are subclassified in P2X and P2Y. Purines modulate many physiological and pathological mechanisms in the CNS. There is a noticeable overlap between social isolation dysfunction and the processes modulated by purinergic signaling. The main goal of this work was verify if post-weaning social isolation induces impairments in purinergic signaling. Thus, we randomly allocated Wistar rats at 21 postnatal day to social rearing or 8 weeks of social isolation. After the 8 weeks, we investigated the behavioral phenotype and performed neurochemical evaluation related to purinergic system. Initially, the results showed that post-weaning social isolation disrupted sensorimotor gating and increased social interaction. Posteriorly, we characterized the purinergic system in this model. There was an accumulation of ADP in cerebrospinal fluid and an increase of ADP hydrolysis in synaptic cleft of hippocampus and striatum of isolation rearing rats. Lastly, we evaluated which purinergic elements might be differentially expressed. To the best of our knowledge, were evidenced several purinergic receptors alteration in this neurodevelopmental model. In the prefrontal cortex we have found an upregulation of adora2a, p2ry1 and p2rx5 genes. Regarding the hippocampus, we demonstrate that p2ry4, p2ry13 and p2ry14 were downregulated. On the other hand, striatum was highly affected and presented several genes downregulated such as adora2a, p2rx4, p2ry2, p2ry6, p2ry12, p2ry13, entpd1, entpd2 and entpd3. Although the neurodevelopmental neuropsychiatric model exhibited slight behavioral impairment, we now provide genetic evidences of several neurobiological processes that were mediated by purinergic signaling. These results may provide a construct validity of several neuropsychiatric conditions. Thus, our results evidenced that social isolation promotes several dysfunctions in the purinergic signaling. Based on the literature results, these alterations in the expression of purinergic receptors indicates a possible impairment in cognition and dopamine and glutamate neurotransmission in the prefrontal cortex. In the hippocampus, few studies comprise the role of these receptors, although may be feasible a microglial dysfunction. Concerning several disturbances showed in the striatum they seem further related with dopamine neurotransmission and microglial dysfunction

Role of P2X7 Receptors in Immune Responses During Neurodegeneration

P2X7 receptors are ion-gated channels activated by ATP. Under pathological conditions, the extensive release of ATP induces sustained P2X7 receptor activation, culminating in induction of proinflammatory pathways with inflammasome assembly and cytokine release. These inflammatory conditions, whether occurring peripherally or in the central nervous system (CNS), increase blood-brain-barrier (BBB) permeability. Besides its well-known involvement in neurodegeneration and neuroinflammation, the P2X7 receptor may induce BBB disruption and chemotaxis of peripheral immune cells to the CNS, resulting in brain parenchyma infiltration. For instance, despite common effects on cytokine release, P2X7 receptor signaling is also associated with metalloproteinase secretion and activation, as well as migration and differentiation of T lymphocytes, monocytes and dendritic cells. Here we highlight that peripheral immune cells mediate the pathogenesis of Multiple Sclerosis and Parkinson’s and Alzheimer’s disease, mainly through T lymphocyte, neutrophil and monocyte infiltration. We propose that P2X7 receptor activation contributes to neurodegenerative disease progression beyond its known effects on the CNS. This review discusses how P2X7 receptor activation mediates responses of peripheral immune cells within the inflamed CNS, as occurring in the aforementioned diseases

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice

Objective: N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. Methods: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. Results: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. Conclusion: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification

Envolvimento do sistema purinérgico no modelo de isolamento social em ratos Wistar adultos

O período do neurodesenvolvimento é caracterizado pela ampla estruturação do sistema nervoso central (SNC) e, portanto, é um momento de grande vulnerabilidade a agentes estressores endógenos e exógenos. Estresses ambientais como infecções durante a gravidez, adversidades na infância e isolamento do convívio social podem prejudicar a maturação do SNC, podendo, em última instância, servir como gatilho para o desenvolvimento de transtornos psiquiátricos. Roedores submetidos ao modelo de isolamento social pós-desmame apresentam prejuízos cognitivos, no filtro sensório-motor, na neurotransmissão de dopamina e de plasticidade sináptica. O sistema purinérgico é mediado pela sinalização extracelular de nucleotídeos e nucleosídeos. Com relação aos nucleotídeos de adenina, o ATP é hidrolisado até adenosina por ação de uma eficiente cadeia enzimática chamada de ectonucleotidases. Os nucleotídeos e nucleosídeos podem atuar nos receptores purinérgicos, que são divididos em receptores P1 e P2. Os receptores P2 são ainda subdivididos em P2X e P2Y. No SNC, as purinas exercem inúmeras funções fisiológicas e patológicas. Há uma evidente sobreposição dos mecanismos disfuncionais apresentados por animais socialmente isolados e pelas modulações que o sistema purinérgico exerce. Levando em consideração tais evidências, a presente dissertação teve como objetivo principal verificar se o isolamento social induz alterações na sinalização purinérgica em diferentes estruturas cerebrais de ratos. Para isso, ratos Wistar de 21 dias foram isolados socialmente por 8 semanas, e em seguida, verificamos diferentes fenótipos comportamentais e parâmetros neuroquímicos relacionados com o sistema purinérgico foram conduzidos. Primeiramente, foi observado prejuízos no filtro sensório-motor e aumento da interação social com ratos desconhecidos nos ratos isolados socialmente. Quanto os parâmetros neuroquímicos, nossos resultados mostraram um acúmulo de ADP no líquido cefalorraquidiano e um aumento da hidrólise do ADP em sinaptossomas de hipocampo e estriado nos animais que foram criados isoladamente. Por último, nas análises de expressão gênica, observamos diferentes alterações em diferentes estruturas cerebrais. O córtex pré-frontal apresentou redução na expressão gênica de adora2a, p2ry1 e p2rx5 nos animais que passaram pelo isolamento social. O hipocampo, por sua vez, apresentou regulação positiva dos genes p2ry4, p2ry13 e p2ry14 nos mesmos animais. Foi observado, ainda, que o estriado foi mais afetado pelo isolamento social, uma vez os animais submetidos ao isolamento apresentaram uma modulação negativa dos genes adora2a, p2rx4, p2ry2, p2ry6, p2ry12, p2ry13, entpd1, entpd2 e entpd3. Desta forma, nossos resultados demonstraram que o isolamento social pós-desmame promove abundantes alterações na sinalização purinérgica. Baseado em dados existentes na literatura, os dados obtidos nesse estudo podem indicar, no córtex pré-frontal, alterações de receptores envolvidos em processos cognitivos e de neurotransmissão de dopamina e glutamato. No hipocampo, apesar da escassez de estudos acerca desses receptores, parece haver indícios de disfunção microglial. O estriado, por sua vez, apresentou alterações de receptores associados, principalmente, com modulação de dopamina e disfunção microglial.The neurodevelopmental period it is characterized by wide structuring of central nervous system (CNS), which implies in highly susceptibility to endogenous or exogenous stressors. Environmental stressor in early-life, such as maternal separation, childhood adversities or social isolation, can disrupt brain development prompting psychiatric diseases. Post-weaning social isolation in rodents causes impairments in cognition, sensorimotor gating, dopamine neurotransmission and synaptic plasticity. The purinergic system is mediated by extracellular signaling of nucleotides and nucleosides. The ATP is hydrolyzed to adenosine by an efficient enzyme chain named as ectonucleotidases. The nucleotides and nucleosides can act on the purinergic receptors, which are divided in P1 and P2 receptors. P2 receptors are subclassified in P2X and P2Y. Purines modulate many physiological and pathological mechanisms in the CNS. There is a noticeable overlap between social isolation dysfunction and the processes modulated by purinergic signaling. The main goal of this work was verify if post-weaning social isolation induces impairments in purinergic signaling. Thus, we randomly allocated Wistar rats at 21 postnatal day to social rearing or 8 weeks of social isolation. After the 8 weeks, we investigated the behavioral phenotype and performed neurochemical evaluation related to purinergic system. Initially, the results showed that post-weaning social isolation disrupted sensorimotor gating and increased social interaction. Posteriorly, we characterized the purinergic system in this model. There was an accumulation of ADP in cerebrospinal fluid and an increase of ADP hydrolysis in synaptic cleft of hippocampus and striatum of isolation rearing rats. Lastly, we evaluated which purinergic elements might be differentially expressed. To the best of our knowledge, were evidenced several purinergic receptors alteration in this neurodevelopmental model. In the prefrontal cortex we have found an upregulation of adora2a, p2ry1 and p2rx5 genes. Regarding the hippocampus, we demonstrate that p2ry4, p2ry13 and p2ry14 were downregulated. On the other hand, striatum was highly affected and presented several genes downregulated such as adora2a, p2rx4, p2ry2, p2ry6, p2ry12, p2ry13, entpd1, entpd2 and entpd3. Although the neurodevelopmental neuropsychiatric model exhibited slight behavioral impairment, we now provide genetic evidences of several neurobiological processes that were mediated by purinergic signaling. These results may provide a construct validity of several neuropsychiatric conditions. Thus, our results evidenced that social isolation promotes several dysfunctions in the purinergic signaling. Based on the literature results, these alterations in the expression of purinergic receptors indicates a possible impairment in cognition and dopamine and glutamate neurotransmission in the prefrontal cortex. In the hippocampus, few studies comprise the role of these receptors, although may be feasible a microglial dysfunction. Concerning several disturbances showed in the striatum they seem further related with dopamine neurotransmission and microglial dysfunction

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice

N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. Methods: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. Results: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. Conclusion: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice

Objective: N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. Methods: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. Results: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. Conclusion: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice

N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. Methods: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. Results: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. Conclusion: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification

Role of P2X7 Receptors in Immune Responses During Neurodegeneration

P2X7 receptors are ion-gated channels activated by ATP. Under pathological conditions, the extensive release of ATP induces sustained P2X7 receptor activation, culminating in induction of proinflammatory pathways with inflammasome assembly and cytokine release. These inflammatory conditions, whether occurring peripherally or in the central nervous system (CNS), increase blood-brain-barrier (BBB) permeability. Besides its well-known involvement in neurodegeneration and neuroinflammation, the P2X7 receptor may induce BBB disruption and chemotaxis of peripheral immune cells to the CNS, resulting in brain parenchyma infiltration. For instance, despite common effects on cytokine release, P2X7 receptor signaling is also associated with metalloproteinase secretion and activation, as well as migration and differentiation of T lymphocytes, monocytes and dendritic cells. Here we highlight that peripheral immune cells mediate the pathogenesis of Multiple Sclerosis and Parkinson’s and Alzheimer’s disease, mainly through T lymphocyte, neutrophil and monocyte infiltration. We propose that P2X7 receptor activation contributes to neurodegenerative disease progression beyond its known effects on the CNS. This review discusses how P2X7 receptor activation mediates responses of peripheral immune cells within the inflamed CNS, as occurring in the aforementioned diseases

Role of P2X7 Receptors in Immune Responses During Neurodegeneration

P2X7 receptors are ion-gated channels activated by ATP. Under pathological conditions, the extensive release of ATP induces sustained P2X7 receptor activation, culminating in induction of proinflammatory pathways with inflammasome assembly and cytokine release. These inflammatory conditions, whether occurring peripherally or in the central nervous system (CNS), increase blood-brain-barrier (BBB) permeability. Besides its well-known involvement in neurodegeneration and neuroinflammation, the P2X7 receptor may induce BBB disruption and chemotaxis of peripheral immune cells to the CNS, resulting in brain parenchyma infiltration. For instance, despite common effects on cytokine release, P2X7 receptor signaling is also associated with metalloproteinase secretion and activation, as well as migration and differentiation of T lymphocytes, monocytes and dendritic cells. Here we highlight that peripheral immune cells mediate the pathogenesis of Multiple Sclerosis and Parkinson’s and Alzheimer’s disease, mainly through T lymphocyte, neutrophil and monocyte infiltration. We propose that P2X7 receptor activation contributes to neurodegenerative disease progression beyond its known effects on the CNS. This review discusses how P2X7 receptor activation mediates responses of peripheral immune cells within the inflamed CNS, as occurring in the aforementioned diseases