Contribution of NPY Y5 Receptors to the Reversible Structural Remodeling of Basolateral Amygdala Dendrites in Male Rats Associated with NPY-Mediated Stress Resilience.

Endogenous neuropeptide Y (NPY) and corticotrophin-releasing factor (CRF) modulate the responses of the basolateral amygdala (BLA) to stress and are associated with the development of stress resilience and vulnerability, respectively. We characterized persistent effects of repeated NPY and CRF treatment on the structure and function of BLA principal neurons in a novel organotypic slice culture (OTC) model of male rat BLA, and examined the contributions of specific NPY receptor subtypes to these neural and behavioral effects. In BLA principal neurons within the OTCs, repeated NPY treatment caused persistent attenuation of excitatory input and induced dendritic hypotrophy via Y5 receptor activation; conversely, CRF increased excitatory input and induced hypertrophy of BLA principal neurons. Repeated treatment of OTCs with NPY followed by an identical treatment with CRF, or vice versa, inhibited or reversed all structural changes in OTCs. These structural responses to NPY or CRF required calcineurin or CaMKII, respectively. Finally, repeated intra-BLA injections of NPY or a Y5 receptor agonist increased social interaction, a validated behavior for anxiety, and recapitulated structural changes in BLA neurons seen in OTCs, while a Y5 receptor antagonist prevented NPY's effects both on behavior and on structure. These results implicate the Y5 receptor in the long-term, anxiolytic-like effects of NPY in the BLA, consistent with an intrinsic role in stress buffering, and highlight a remarkable mechanism by which BLA neurons may adapt to different levels of stress. Moreover, BLA OTCs offer a robust model to study mechanisms associated with resilience and vulnerability to stress in BLA.SIGNIFICANCE STATEMENT Within the basolateral amygdala (BLA), neuropeptide Y (NPY) is associated with buffering the neural stress response induced by corticotropin releasing factor, and promoting stress resilience. We used a novel organotypic slice culture model of BLA, complemented with in vivo studies, to examine the cellular mechanisms associated with the actions of NPY. In organotypic slice cultures, repeated NPY treatment reduces the complexity of the dendritic extent of anxiogenic BLA principal neurons, making them less excitable. NPY, via activation of Y5 receptors, additionally inhibits and reverses the increases in dendritic extent and excitability induced by the stress hormone, corticotropin releasing factor. This NPY-mediated neuroplasticity indicates that resilience or vulnerability to stress may thus involve neuropeptide-mediated dendritic remodeling in BLA principal neurons.

NPY Induces Stress Resilience via Downregulation of Ih in Principal Neurons of Rat Basolateral Amygdala.

Neuropeptide Y (NPY) expression is tightly linked with the development of stress resilience in rodents and humans. Local NPY injections targeting the basolateral amygdala (BLA) produce long-term behavioral stress resilience in male rats via an unknown mechanism. Previously, we showed that activation of NPY Y1 receptors hyperpolarizes BLA principal neurons (PNs) through inhibition of the hyperpolarization-activated, depolarizing H-current, Ih The present studies tested whether NPY treatment induces stress resilience by modulating Ih NPY (10 pmol) was delivered daily for 5 d bilaterally into the BLA to induce resilience; thereafter, the electrophysiological properties of PNs and the expression of Ih in the BLA were characterized. As reported previously, increases in social interaction (SI) times persisted weeks after completion of NPY administration. In vitro intracellular recordings showed that repeated intra-BLA NPY injections resulted in hyperpolarization of BLA PNs at 2 weeks (2W) and 4 weeks (4W) after NPY treatment. At 2W, spontaneous IPSC frequencies were increased, whereas at 4W, resting Ih was markedly reduced and accompanied by decreased levels of HCN1 mRNA and protein expression in BLA. Knock-down of HCN1 channels in the BLA with targeted delivery of lentivirus containing HCN1-shRNA increased SI beginning 2W after injection and induced stress resilience. NPY treatment induced sequential, complementary changes in the inputs to BLA PNs and their postsynaptic properties that reduce excitability, a mechanism that contributes to less anxious behavior. Furthermore, HCN1 knock-down mimicked the increases in SI and stress resilience observed with NPY, indicating the importance of Ih in stress-related behavior.SIGNIFICANCE STATEMENT Resilience improves mental health outcomes in response to adverse situations. Neuropeptide Y (NPY) is associated with decreased stress responses and the expression of resilience in rodents and humans. Single or repeated injections of NPY into the basolateral amygdala (BLA) buffer negative behavioral effects of stress and induce resilience in rats, respectively. Here, we demonstrate that repeated administration of NPY into the BLA unfolds several cellular mechanisms that decrease the activity of pyramidal output neurons. One key mechanism is a reduction in levels of the excitatory ion channel HCN1. Moreover, shRNA knock-down of HCN1 expression in BLA recapitulates some of the actions of NPY and causes potent resilience to stress, indicating that this channel may be a possible target for therapy.

Estudio del sistema purinérgico a través de la caracterización de la enzima E-NPP3 y de la secreción de ATP en células uroepiteliales de la vejiga urinaria del ratón / Study of the purinergic system through the characterization of the E-NPP3 enzyme and the secretion of ATP in uroepithelial cells of the urinary bladder of the mouse

El presente trabajo involucra el estudio del sistema purinérgico mediante la purinérgico mediante la purificación y la caracterización bioquimica de la ecto-enzima E-NPP3 soluble, la cual modula la activación de los receptores purinérgicos mediante la hidrólisis de los nucleótidos extracelulares. La purificación de esta enzima, expresada en la línea celular CHO-K1, requirió el empleo de diferentes columnas cromatográficas; comprobándose la pureza, mediante la determinación de la actividad enzimática, la cuantificación de las proteínas y la detección de dicha enzima. Los resultados de la caracterización de la E-NPP3 indican que presentan un ph óptimo alcalino y que su actividad depende de la la concentración de los iones calcio y magnesio: mientras que el imidazol y el DDT ejercen acciones inhibidoras sobre su actividad. La purificación de la enzima E-NPP3 soluble representa un primer paso para futuros estudios que permitan su cristalización lo cual, constituirá una herramienta en la elaboración de agonistas y antagonistas selectivos o de anticuerpos monoclonales contra dicha enzima, útiles en el diagnóstico o el tratamiento de condiciones patológicas como el cáncer de colon y la colangiocarcinoma. Adicionalmente, teniendo en cuenta que se ha demostrado la disminución de la expresión de las ecto-nucleotidasas en el uroepitelio de pacientes que padecen cistitis intersticial; el presente trabajo comprende también, el desarrollo de un modelo de órgano aislado, la vejiga urinaria del ratón, para el estudio de la secreción de ATP desde las células uroepiteliales. Dicho modelo permitió demostrar, mediante estudios electrofisiológicos y el uso de diferentes fármacos, la contriución de los receptores purinérgicos sobre la actividad eléctrica del nervio pélvico cuando la vejiga es sometida a distensión mecánica gradual; sugiriendo la importancia de estos receptores en la transducción mecanosensorial de dicho órgano y permitiendo inferir la posible interrelación con los receptores de vaniloides en la detección de los estímulos sensoriales por parte del uroepitelio