Organotypic Basolateral Amygdala Slice Cultures: A Model for Stress-related Circuitry

• Author / Creator

  Michaelson, Sheldon D

• Stress generates an adaptive behavioral and physiological response that prepares an organism for new environmental pressures. However, if stressors are perceived as uncontrollable, prolonged, or severe, they can produce exaggerated responses and result in neuropsychiatric syndromes such as anxiety and depression. Many brain regions respond to stress with the basolateral amygdala (BLA) regulating emotional processing. Glutamatergic principal cells are the major output neurons of the BLA and mediate behavioral stress responses. These cells respond to stress with dendritic hypertrophy and increased spine densities. Several endogenous neuromodulators alter BLA output cell activity, including the anxiolytic neuropeptide Y (NPY) and the anxiogenic corticotrophin releasing factor (CRF). Daily (5 x d) injections of NPY or urocortin (CRF-R1 and CRF-R2 agonist) induce a persistent state of stress resilience or vulnerability, respectively. While the mechanisms governing the acute actions of NPY and CRF on the stress response in the amygdala are well established, the signal transduction pathways mediating NPY- and CRF-induced stress resilience and vulnerability are unknown. We hypothesized that chronic CRF treatment would recapitulate the effects of stress observed on BLA principal cells and NPY treatment would result in the opposite effect, namely, dendritic hypotrophy. We tested this in male rats in vivo and using a novel organotypic slice culture preparation of BLA (BLA OTCs) we recently developed, optimized and validated. BLA OTCs (6 w postnatal equivalent age) were treated with varying concentrations of NPY, CRF, NPY prior to CRF, CRF prior to NPY, NPYreceptor- subtype-selective agonists, NPY + protein phosphatase inhibitors or CRF + kinase inhibitor for 5 days. Electrophysiological and morphological changes in neurobiotin-filled neurons were analyzed 7-14 days after peptide treatment. iii In BLA OTCs, NPY, and the Y5-agonist, but not the Y1-agonist, decreased excitatory drive onto principal cell, while also reducing cell capacitance and culminated in dendritic hypotrophy as determined by Sholl analysis. CRF and the anxiogenic Y2-agonist produced opposite effects to those same neuronal properties observed with NPY and the Y5-agonist, and similar to that seen with stress. The BLA OTC preparation thus may predict structural changes that accompany stress resilience and vulnerability. NPY and CRF cause opposite effects on BLA dendrites correlating with behavioral changes seen with peptide treatment in vivo, which can be prevented when incubated with the opposing neuropeptide. Moreover, NPY- and CRF-induced structural plasticity is regulated by the opposing actions of the protein phosphatase calcineurin and the protein kinase Ca2+/calmodulin-dependent protein kinase II, respectively. To confirm our finding and further validate the BLA OTC model, NPY, Y1- agonist or a Y5-agonist were injected daily (5 x d) into the BLA of 7 week-old rats; social interaction (SI) was assessed and similar recordings were made in acute BLA slices 4 weeks after the first injection. NPY, and the Y5-agonist increased SI time at all experimental time points studied (up to 4 weeks). Intra-BLA injections of the Y1-agonist increased SI on days 1 and 5 after injection but did not affect SI long-term. BLA principal cell dendrites were retracted in NPY- and Y5-agonist-treated, but not Y1 agonist-treated rats. These studies indicate a novel structural correlate of stress resilience and vulnerability, while demonstrating the potency of the BLA OTC model for studying the underlying cellular and molecular mechanisms of stress/anxiety-related behavior.

• Subjects / Keywords

  • Neuropeptide Y
  • Stress and anxiety
  • Amygdala

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3PR7N84P

• 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.