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Review
. 2017 Sep 1;178:43-65.
doi: 10.1016/j.physbeh.2016.11.014. Epub 2016 Nov 18.

A Users Guide to HPA Axis Research

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Free PMC article
Review

A Users Guide to HPA Axis Research

Robert L Spencer et al. Physiol Behav. .
Free PMC article

Abstract

Glucocorticoid hormones (cortisol and corticosterone - CORT) are the effector hormones of the hypothalamic-pituitary-adrenal (HPA) axis neuroendocrine system. CORT is a systemic intercellular signal whose level predictably varies with time of day and dynamically increases with environmental and psychological stressors. This hormonal signal is utilized by virtually every cell and physiological system of the body to optimize performance according to circadian, environmental and physiological demands. Disturbances in normal HPA axis activity profiles are associated with a wide variety of physiological and mental health disorders. Despite numerous studies to date that have identified molecular, cellular and systems-level glucocorticoid actions, new glucocorticoid actions and clinical status associations continue to be revealed at a brisk pace in the scientific literature. However, the breadth of investigators working in this area poses distinct challenges in ensuring common practices across investigators, and a full appreciation for the complexity of a system that is often reduced to a single dependent measure. This Users Guide is intended to provide a fundamental overview of conceptual, technical and practical knowledge that will assist individuals who engage in and evaluate HPA axis research. We begin with examination of the anatomical and hormonal components of the HPA axis and their physiological range of operation. We then examine strategies and best practices for systematic manipulation and accurate measurement of HPA axis activity. We feature use of experimental methods that will assist with better understanding of CORT's physiological actions, especially as those actions impact subsequent brain function. This research approach is instrumental for determining the mechanisms by which alterations of HPA axis function may contribute to pathophysiology.

Keywords: ACTH; Corticosterone; Cortisol; Glucocorticoid; HPA axis; Stress.

Figures

Figure 1
Figure 1
HPA axis and endogenous glucocorticoid hormones (CORT). A. The HPA Axis consists of 3 populations of cells and the specialized hormones that each secretes: 1] neurons in the medial parvocellular portion of the hypothalamic paraventricular nucleus (PVN) secrete corticotropin releasing hormone (CRH), 2] endocrine cells (corticotrophs) in the anterior pituitary secrete adrenocorticotropic hormone (ACTH), and 3] endocrine cells primarily in the zona fasciculata of the adrenal cortex secrete the glucocorticoid hormones cortisol and/or corticosterone (CORT). CORT is secreted into the systemic circulation [4] and affects cells throughout the body, including the brain. CORT produces direct negative feedback inhibition of corticotrophs in the anterior pituitary [5] and CRH neurons in the PVN [6]. Activity of the HPA axis is directly and indirectly controlled by various neural activity present throughout the forebrain and brainstem [7]. Direct innervation of the CRH neurons in the PVN includes afferents from the bed nucleus of the stria terminalis (BNST), the dorsomedial hypothalamus (DMH), the ventromedial hypothalamus (VMH), the nucleus tractus solitarius (NTS), and the ventrolateral medulla (VLM). Indirect control includes the medial prefrontal cortex (mPFC), hippocampus (HC), amygdala (Amyg) and septum. B. The principal endogenous glucocorticoid hormones in vertebrates are the closely related steroid molecules (4 carbon ring based structure) cortisol and corticosterone.
Figure 2
Figure 2
Basic structure of CRF and ACTH hormones. Diagrams illustrate the basic precursor protein structure and the final cleavage product peptide hormone amino acid sequence (single letter code) for (A) corticotropin releasing factor (also known as corticotropin releasing hormone) and (B) adrenocorticopic hormone found in human, mouse and rat.
Figure 3
Figure 3
Idealized basal and stress-induced CORT secretion pattern in the rat. A. Basal corticosterone (CORT) secretion in the rat has an ultradian (hourly pulses) and circadian rhythm. The basal peak CORT secretion of this nocturnal species coincides with the beginning of the rat’s active period. Relative ultradian and circadian basal cortisol levels are similar in humans except that peak basal levels occur at the opposite time of day (around dawn). B. Stimulation of the HPA axis, such as occurs with acute stress, produces similar maximal CORT levels regardless of the time of day. Red horizontal lines above both acute stress CORT responses depict the time of stressor onset and duration. Typically CORT levels reach their maximum within 30 min after stressor onset, and return to basal levels 60–90 min after stressor termination.
Figure 4
Figure 4
Representative ACTH and CORT plasma levels present in rats 30 min after onset of various stressors. A. Blood samples (tail clip method) of adult male Sprague-Dawley rats were collected immediately after 30 min of first time exposure to a housing tub lacking bedding, circular arena, elevated pedestal or restraint tube. Control samples were collected at the same time of day from rats that had been left undisturbed in their home cage. *significantly different from control levels (p < 0.05) (adapted from (82)). B. Blood samples (trunk blood) of adult male Sprague-Dawley rats were collected immediately after 30 min of first time exposure to restraint tube, forced swim at 25 °C, or intermittent footshock. Control samples were collected at the same time of day from rats that had been left undisturbed in their home cage. *significantly different from control levels (p < 0.05); †significantly different from restraint (p < 0.05) (adapted from (83)).
Figure 5
Figure 5
Time-course for increased plasma CORT levels in 3 rats during restraint. Rats (adult male Sprague-Dawley) were placed in a restraint tube within 30 seconds after experimenter entry into the animal housing room, and serial blood samples (~50 μl/sample) were collected from the tail vein (tail clip method) (RL Spencer, previously unpublished data).
Figure 6
Figure 6
Rat strain comparison of CORT response to long session of restraint (4 hr) followed immediately by novel stressor challenge. Adult male rats of 3 different rat strains (Fischer 344 – F344, Sprague-Dawley – SD, and Lewis – LEW) were exposed to restraint (Plexiglas tube) for 4 h, and were then immediately exposed to a different novel stressor (wire mesh tube restraint) for an additional 15 min. Serial blood samples were collected from the tail vein (tail clip method). †significantly different from baseline (0 h) (p < 0.05), §significantly different from 4 hr time-point for the same strain (p < 0.05), *significantly different from corresponding SD and LEW value (p < 0.05). (reprinted from (81))
Figure 7
Figure 7
Plasma CORT levels produced by systemic injection of rats with different CORT doses. A. Plasma CORT levels in serial blood samples (tail clip method) from adrenalectomized (ADX) adult male Sprague-Dawley rats after injection with 3 different doses of corticosterone (1.25, 2.5 or 5.0 mg/kg, i.p.) (reprinted from (186)). B. Plasma CORT levels in serial blood samples (tail clip method) from adrenal-intact adult male Sprague-Dawley rats after injection with 3 different doses of corticosterone (1.25, 2.5 or 3.75 mg/kg, s.c.) or vehicle (16% EtOH, 44% propylene glycol, 40% phosphate buffer saline, s.c.) (reprinted from (187)).
Figure 8
Figure 8
CORT (2.5 mg/kg s.c.) produces hippocampal free CORT levels comparable to that seen after footshock stress. After collection of two baseline samples using microdialysis probes targeting the hippocampus, adult male Sprague-Dawley rats (n=3–4 per group) were exposed to 80 footshocks (1.0 mA, 5 secs each, 90 sec variable ITI) or were injected with 2.5 mg/kg (s.c.) and placed into a standard microdialysis bowl. Sampling interval was 30 min epochs and perfusion rate was 2 μl/min. Corticosterone was measured in dialysates utilizing standard RIA procedures. Peak concentrations of CORT in dialysates were identical between experimental groups, although CORT-treated rats exhibited an earlier peak (T Deak, previously unpublished data).
Figure 9
Figure 9
CORT in drinking water restores daily basal peak in circulating CORT levels, but with some time-lag compared to endogenous CORT secretion. Plasma CORT levels in serial blood samples (jugular catheter automated blood sampling system) from adrenalectomized (ADX) or adrenal-intact (Sham-ADX) adult male Sprague-Dawely rats over the course of 2 days (solid black bar above x-axis indicates dark phase). ADX rats had CORT in the drinking water (25 μg/ml in 0.9% saline containing 0.45% hydroxyl-propyl-beta-cyclodextrin) for the first day shown. Between-group comparisons: *p < 0.05, **p < 0.01, ***p < 0.001. (adapted from data presented in (203), courtesy of CA Lowry).

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