| Stress is encountered on a daily basis. While most stress may not be life threatening per se, excessive amounts can lead to dysregulated stress responses that normally serve an adaptive role to maintain optimal performance under daily pressures. This can lead to, among other things, elevated levels of anxiety and fear and/or expression of fear in response to nonthreatening stimuli, and can ultimately manifest as anxiety-and stress-related disorders. Understanding the circuits activated to buffer stress responses will provide insight into management strategies for the treatment of anxiety disorders, identify factors that predispose individuals to these disorders, and determine why some individuals are better able to cope with stress. The basolateral amygdaloid complex (BLA) serves as a critical region for overseeing appropriate autonomic, endocrine, and behavioral responses to stress. Neuropeptide Y (NPY) acts within the BLA as an anxiolytic or anxiety-reducing compound, whose role was recently expanded to include the ability to produce stress resilience and long-lasting reductions in anxiety-like behavior via activation of the phosphatase, calcineurin (CaN). Although the receptor subtypes mediating the behavior effects of NPY in the BLA have been well-characterized, the origin of NPY input to the BLA and the signaling molecules and cellular substrates mediating NPY's effects are poorly defined. The initial part of this thesis mainly focused on: 1) identifying brain regions that send NPY projections to the BLA using retrograde tract tracing coupled with immunohistochemistry (IHC) and 2) determining which of these projections might be involved in regulating anxiety and fear by assessing changes in NPY expression following presentation of a conditioned fearful stimulus. Tracing studies revealed novel sources of NPY input to the BLA, most notably from the amygdalostriatal transition area (AStr) and stria terminalis. Shortly after testing expression of conditioned contextual fear, conditioned rats had lower immunoreactive (ir) fiber intensities for the amidated, releasable form of NPY in the BLA, suggesting that NPY was likely released into the synapse. Furthermore, using in situ hybridization coupled with emulsion autoradiography, it was demonstrated that conditioned rats had higher NPY mRNA within AStr cells, but not within the BLA and stria terminalis. Together, these studies suggest that NPY projections from the AStr to the BLA may regulate anxiety and fear responses to conditioned stimuli. The final part of this thesis focused on proposing a model for how NPY could be producing its persistent anxiolytic and stress resilient effects. These studies assessed CaN colocalization with BLA neuronal cell-types and the NPY Y1 receptor (Y1r). Additionally, using a model of NPY-induced stress resilience, in which repeated injections of NPY were performed in the BLA, long-term changes in BLA spine density were assessed using IHC for the well-characterized spine marker, spinophilin. Studies revealed that CaN immunoreactivity was primarily restricted to pyramidal neurons in the BLA, and that these neurons exhibited a large degree of colocalization of CaN and Y1r immunoreactivity. This indicates that regulation of CaN by NPY may occur by activation of Y1r located on BLA pyramidal neurons. Rats treated with NPY displayed a reduction in anxiety-like behavior as assessed by the social interaction test and a long-term reduction in spinophilin-immunoreactive punctae density in the lateral amygdaloid nuclear division of the BLA in comparison to vehicle-injected controls. Since spines within the BLA are largely restricted to pyramidal neurons and spines receive the great proportion of excitatory synaptic input onto the cell, these results suggest NPY treatment caused a reduction in spines and potentially excitatory synaptic input onto LA pyramidal neurons. Ultimately, this could provide a structural basis for the persistent anxiety-reducing properties of NPY in the BLA. Overall, the studies described in this thesis provide exciting new targets for assessing NPY regulation of BLA function, with which further characterization may lead to a better understanding of individual differences in stress reactivity as well as alternative pharmacotherapies for anxiety-related disorders. |
