Long-Lived Organotypic Slice Culture Model of the Rat Basolateral Amygdala.

Organotypic slice cultures (OTCs) have been employed in the laboratory since the early 1980s and have proved to be useful for the study of a number of neural systems. Our recent work focuses on the development of behavioral stress resilience induced by repeated daily injections of neuropeptide Y into the basolateral amygdala (BLA). Resilience develops over weeks, persisting to 8 weeks. To unravel the cellular mechanisms underlying neuropeptide Y-induced stress resilience we developed in vitro OTCs of the BLA. Here, we provide an optimized protocol that consistently yields viable and healthy OTCs containing the BLA and surrounding tissue using the interface method, prepared with slices taken from postnatal (P) day 14 rats. We explain key points to optimizing tissue viability and discuss mitigation or avoidance of pitfalls that can arise to aid in successful implementation of this technique. We show that principal neurons in BLA OTCs (8 weeks in vitro = equivalent postnatal day 70) develop into networks that are electrophysiologically very similar to those from acute slices obtained from older rats (P70) and respond to pharmacological treatments in a comparable way. Furthermore, we highlight how these cultures be used to further understand the molecular, cellular, and circuit-level neuropathophysiological changes underlying stress disorders. BLA OTCs provide long-term physiological and pharmacological results whose predictions were borne out in vivo, supporting the validity of the BLA OTC as a model to unravel BLA neurocircuitry. Recent preliminary results also support the successful application of this approach to preparing long-lived OTCs of BLA and neocortex from mice. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Organotypic slice culture Support Protocol 1: Changing medium Support Protocol 2: Drug incubations Basic Protocol 2: Excision of OTC slices from inserts Support Protocol 3: Fixation of slices.

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.