Mechanisms of NPY Y2 Receptor-Mediated Anxiogenesis in the Basolateral Amygdala

  • Author / Creator
    Mackay, James P
  • Fear and anxiety are highly adaptive emotions that motivate species appropriate responses to threats: immediate and potential. Certain threats, such as ancestral predators, are highly predictable and can be recognized and addressed with innate (inherited) neural systems. This “genetic memory” underlies human phobias (ex. fear of snakes) and allows individuals to recognize and effectively respond to conserved dangers, regardless of previous encounters. Unlearned fear systems, however, have clear limitations, the most fundamental being their inability to recognize every conceivable danger. Plastic threat appraisal systems overcome this limitation. The basolateral amygdala (BLA) is the principal brain site where learned associations between innocuous sensory cues and intrinsically aversive stimuli are formed (fear conditioning). Fear conditioning is though to model a key mechanism for identifying novel threats. Anxiety, a more sustained state of hyper-vigilance, is also mediated by the BLA and is most appropriate when threats are diffuse and not readily predicted by explicit cues. In susceptible individuals, exposure to a severe unpredictable stressor can elicit a prolonged disordered anxiety state termed posttraumatic stress disorder (PTSD). Although protective to a degree, PTSD substantially impairs normal functioning and is profoundly unpleasant for the sufferer. An important factor thought to protect some individuals from the deleterious effects of traumatic stress is Neuropeptide Y (NPY). BLA NPY infusions are highly anxiolytic in rodents, while repeated infusions produce plastic changes culminating in a long lasting low-anxiety state. Glutamatergic Principal neurons (PN) are the BLA’s majority neuron type (85%) and mediate its output. The remaining 15% are a diverse group of GABA interneurons that tightly regulate PN activity. The output of a population PNs signals fear and anxiety. We previously showed that NPY inhibits BLA PNs via postsynaptic NPY Y1 receptors (consistent with anxiolytic actions). Furthermore, Y1 selective agonists mimic NPYs acute in vivo anxiolytic effects. Surprisingly, selective activation of BLA Y2 receptors (Y2-R) increases anxiety by an (until now) unknown mechanism. The principal focus of this thesis is to mechanistically dissect Y2-R functions in the BLA. A secondary aim is to determine if and how Y2-Rs contribute to the overall anxiolytic actions of the full agonist NPY. Y2-Rs are typically presynaptic and inhibit neurotransmitter release. We therefore, hypothesized Y2-Rs disinhibit PNs by decreasing BLA interneuron GABA release. To test this, we used slice-patch electrophysiology in rat BLA-containing brain slices. Application of the selective Y2-R agonist [ahx5-24]NPY decreased the frequency of PN miniature GABAA inhibitory postsynaptic currents (IPSC)s with no effect on amplitude (suggesting a presynaptic effect). Interestingly, in the absence of tetrodotoxin (TTX) [ahx5-24]NPY increased the frequency of large amplitude fast kinetic sIPSCs, suggesting disinhibition of another interneuron type. To determine the Y2-R expressing interneuron type mediating the above effects, we used a mouse model engineered to express the TdTomato fluorophore under control of the Y2-R gene promoter. Immunohistochemistry studies suggested Y2-Rs are expressed exclusively on interneurons characterized by NPY and somatostatin (SOM) expression. SOM interneurons innervate PN dendrites and also target other interneuron types consistent with electrophysiology findings. In addition to the above effects, [ahx5-24]NPY increased PN excitability (indicated by a decrease in the depolarizing current required to elicit action potentials). These findings are consistent with the in vivo anxiogenic effects of selective Y2-R agonists. Although [ahx5-24]NPY only slightly depolarized most PNs, it substantially increased PN input resistance, indicating a net closure of ion channels. We hypothesized these effects were due to reduced tonic GABAA-mediated inhibition. However voltage-clamp experiments indicated [ahx5-24]NPY reduced a PN G-protein coupled inward rectifying K+ conductance (GIRK). Subsequent experiments revealed this was due to reduced tonic GABAB-R activation. Since PNs express postsynaptic GABAB-Rs exclusively at their dendrites this effect is also consistent with actions on (Y2-R expressing) NPY/SOM interneurons. Surprisingly, this Y2-R action persisted in TTX, indicating it is largely action potential-independent. Ultimately this finding reveals a highly novel consequence of action potential-independent neurotransmission. NPY-mediated plasticity requires the Ca2+-dependent phosphatase calcineurin (which mediates LTD-type learning and is expressed in dendrites). Dendritic GABAB-GIRKs facilitate the Mg2+ block of NMDA receptors and dampen plasticity. Thus Y2-Rs may function to disinhibit PN dendrites and facilitate Ca2+-dependent, NPY-mediated plasticity. [ahx5-24]NPY-mediated increases in PN calcium influx were revealed indirectly by an increase Ca2+ dependent slow after-hyperpolarization (sIAHP) K+ current in half of all responsive PNs.

  • Subjects / Keywords
  • Graduation date
    Spring 2016
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.