NPY‐Induced Stress Resilience and Downregulation of HCN1 Channels Modifies Expression of Conditioned Fear

Abstract

The anxiolytic and stress‐buffering properties of neuropeptide Y (NPY) in the basolateral amygdala (BLA) have long been established in animal models as well as in humans. Repeated injections of NPY into the BLA promote long‐lasting resilience to restraint stress (Sadjyk et al., 2008), which is associated with downregulation of HCN1 channels. In addition to inducing stress resilience, we hypothesize that repeated injections of NPY and downregulation of HCN1 subunits in the BLA will ameliorate freezing behavior in a cued fear conditioning paradigm. Male rats received bilateral infusions of 10pmol NPY or vehicle (100nl; 0.1M PBS) into the BLA repeated daily for 5 days. Two weeks after the injections, animals underwent a cued fear conditioning protocol: On day 1, animals were placed in chamber 1 and received 4 pairings of a tone (1500Hz, 85dB) with a foot shock (0.4mA; fear acquisition). The following day the rats were placed in novel chamber 2 and received 15 trials of the tone without a foot shock at 60s intertrial intervals (fear extinction) and freezing behavior was monitored (Anymaze software). Both vehicle‐ and NPY‐treated rats displayed similar rates and amplitudes of fear acquisition on day 1. On day 2, NPY‐treated rats exhibited significantly lower freezing in the first five sessions of the extinction phase. Additionally, at the time point we performed the fear conditioning experiment, we observed an induction of LC3B expression (an autophagy marker) and downregulation of brain‐derived nerve factor (BDNF), however downregulation of the HCN1 protein is not seen until 4 weeks after the NPY treatment. Lastly, to test the involvement of HCN1 subunits in fear conditioning, HCN1 protein was knocked‐down in the BLA using lentiviral delivery of HCN1‐shRNA. Four weeks after the surgery, the rats underwent the same cued fear conditioning protocol as described above and freezing behavior was monitored. HCN1‐knockdown animals had reduced freezing on the acquisition day. This work indicates that the induction and maintenance of NPY‐induced stress resilience is associated with differential changes in protein expression. NPY‐induced resilience is correlated with BDNF downregulation and induction of autophagic pathways at early time points, while HCN1 downregulation later consolidates a new stress‐relieving baseline in NPY‐treated animals. These findings can be of great importance for the way anxiety disorders like post‐traumatic stress disorder are viewed and treated.Support or Funding InformationWork supported by MH090292 (JHU; WFC)