Contribution of NPY Y₅Receptors to the Reversible Structural Remodeling of Basolateral Amygdala Dendrites in Male Rats Associated with NPY-Mediated Stress Resilience

Abstract

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.

Long‐Lived Organotypic Slice Culture Model of the Rat Basolateral Amygdala

“…Together the results here and in our previous publication (Michaelson et al, 2020) clearly validate numerous critical similarities of long-term BLA OTCs with their in vivo counterparts, and directly predict unanticipated and surprising findings in vivo. This makes OTCs of the BLA a potentially very attractive tool with which to study stress-related circuitry and related neuroplasticity.…”

“…Total dendritic length and number of branch points (an index of dendritic complexity) differed between the populations studied (Michaelson et al, 2020;and methods above). BLA OTC PNs at EP70 are considerably smaller than neurons from either P14 or P70 ex vivo slices (we did not analyze soma size due potential errors incurred detaching the pipet from patched neurons).…”

“…In healthy OTCs (EP70), PN properties differ relative to acute ex vivo P14 and P70 cells in the three preparations. Neurons from the three populations were filled with neurobiotin via pipet after electrophysiological characterization, then slices were fixed, stained, cleared, and optically sectioned by confocal microscopy (Michaelson et al, 2020;Silveira Villarroel et al, 2018). (Reagents used for immunocytochemistry illustrated below are provided in the legend to Fig.…”

“…When all electrophysiological recordings have been completed for a given slice, the slice is fixed for staining and imaging. In our experiments, this was an essential step for correlating changes in whole-cell capacitance with changes in morphology caused by repeated treatment with NPY or CRF (Michaelson et al, 2020). We initially followed a fixation protocol that used 10% formalin, but subsequently determined that use of 4% paraformaldehyde significantly improved results.…”

“…These data have been reported in some detail (Michaelson et al, 2020), so comparisons are summarized here; interested readers should consult the cited paper for more details. In brief, PNs in BLA OTCs mature in culture to develop electrophysiological attributes that are distinct from those of PNs in acute ex vivo BLA slices from P14 and P70 rats, which in turn differ from one another.…”