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 Y receptors hyperpolarizes BLA principal neurons (PNs) through inhibition of the hyperpolarization-activated, depolarizing H-current, The present studies tested whether NPY treatment induces stress resilience by modulating 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 in the BLA were characterized. As reported previously, increases in social interaction (SI) times persisted weeks after completion of NPY administration. 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 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 in stress-related behavior. 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.

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 Y 5 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 Y 5 receptor agonist increased social interaction, a validated behavior for anxiety, and recapitulated structural changes in BLA neurons seen in OTCs, while a Y 5 receptor antagonist prevented NPY's effects both on behavior and on structure. These results implicate the Y 5 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.

23Basolateral amygdala (BLA) responses to stress are regulated by endogenous neuropeptide 24 Y (NPY) and corticotrophin-releasing factor (CRF), associated respectively with resilience and 25 vulnerability to stress. We studied mechanisms underlying effects of repeated NPY and CRF 26 treatment on structure and function of BLA principal neurons (PN) in organotypic slice cultures 27 (OTC) of rat BLA. NPY attenuated excitatory input and induced dendritic hypotrophy via Y5-28 receptors, while CRF increased excitatory input and induced hypertrophy of BLA PNs. Structural 29 responses to NPY and CRF required calcineurin and CaMKII, respectively. Finally, repeated in 30 vivo treatment of BLA with NPY or a Y5 receptor agonist increased social interaction and 31 recapitulated structural changes in BLA neurons seen in OTCs. These results implicate the Y5 32 receptor in the long-term, anxiolytic-like effects of NPY in the BLA. Moreover, BLA OTCs offer 33 a robust model to study mechanisms associated with resilience and vulnerability to stress in BLA. 34 35

Current ProtocolsCurrent Protocols Preparation: Day of dissection and slicing 11. Collect vibratome blades from storage, immediately wash off acetone with water, and air dry. Alcohol sterilize and air dry shortly before use.12. Cut off the bottom of a 50-ml Falcon tube at the 35-ml mark to utilize as a chamber for the agarose brain block.13. Prepare a fresh tube of 2% (w/v) low-melting-point agarose in slicing solution as in step 6 and place in the 42°C water bath.This will be enough for two animals and should remain liquid in the tube until needed for the second animal.14. Turn on the laminar flow cabinet and clean the surface using 70% ethanol. Sterilize the hood with UV irradiation for ∼20 min prior to use.15. Clear a workspace on a bench near the hood to use for decapitation. Set up guillotine and surgical scissors.16. Sterilize and place the cyanoacrylate glue, 100-mm plastic Petri dish, 100-mm filter paper, three 24-well plates, ice bucket with ice, the cut Falcon tube top with lid (step 12), two additional 50-ml Falcon tubes, vibratome razor blade, small straight dissecting scissors, straight fine forceps, polished microspatula, industrial razor blade, 1-ml syringe with 25-G, 5/8-in. needle, sable paint brush, paper towel, 25-ml serological pipet with controller, and 1000-μl pipet tips and pipetter in the sterilized biosafety cabinet.Again, all items must be sterilized before being placed in the hood.