draft...draft canadian journal of physiology and pharmacology april 2019 effect of chronic...

Draft

Effect of chronic corticosterone treatment on expression and distribution of serotonin 5-HT7 receptors in rat adrenal

glands

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2019-0080.R1

Manuscript Type: Article

Date Submitted by the Author: 25-Apr-2019

Complete List of Authors: Saroj, Neeshu; CINVESTAV IPN, PharmacologyShanker, Shiv; Instituto Politécnico Nacional Escuela Superior de Medicina, Sección de Estudios de Posgrado e InvestigaciónFernández-Parrilla, Manuel; CINVESTAV IPN, Fisiología, Biofísica y NeurocienciasLopez-Sanchez, Pedro; Instituto Politécnico Nacional Escuela Superior de Medicina, Sección de Estudios de Posgrado e InvestigaciónTerrón, José; CINVESTAV IPN, Pharmacology

Is the invited manuscript for consideration in a Special

Issue:Not applicable (regular submission)

Keyword: ACTH, adrenal cortex, chronic corticosterone treatment, chronic stress, 5-HT7 receptors

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

Draft

Canadian Journal of Physiology and Pharmacology April 2019

Effect of chronic corticosterone treatment on expression and distribution of serotonin 5-HT7

receptors in rat adrenal glands

Neeshu Saroj*,1 Shiv Shanker*,2 Manuel A. Fernández-Parilla,3 Pedro López-Sánchez 2

and José A. Terrón.1

1 Departamento de Farmacología, CINVESTAV-IPN, Av. Instituto Politécnico Nacional 2508,

col. La Laguna Ticomán, CP 07360, CDMX, MEXICO.

2 Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina-IPN, Plan de

San Luis y Díaz Mirón s/n, Casco de Sto Tomás, Ciudad de México, Mexico.

3 Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV-IPN, Av. Instituto

Politécnico Nacional 2508, col. La Laguna Ticomán, CP 07360, CDMX, MEXICO.

* These authors equally contributed to this work

Author for correspondence:

José A. Terrón, Ph.D., at the above address.

Telephone: (52) (55) 5747-3800 ext. 5437

Fax: (52) (55) 5747-3394

e-mail: [email protected]; [email protected]

Page 1 of 30



Draft

2

Abstract

Sensitized stress-induced corticosterone (CORT) secretion in chronically stressed rats involves 5-

HT7 receptor activation. The effect of 14-day chronic CORT and vehicle (VEH) administration

on 5-HT7 receptor expression in adrenal glands, adrenal 5-HT content, and ACTH and CORT

secretion was analysed. On day 15, VEH- and CORT-treated animals were perfused or

decapitated without stress exposure (0 min) or after 10 and 30 min of restraint for collection of

trunk blood and tissues. 5-HT7 receptor-like immunoreactivity (5-HT7R-LI), 5-HT7 receptor

protein and mRNA levels were determined by immunohistochemistry, Western blot and reverse

transcription polymerase chain reaction assays, respectively; 5-HT levels and hormones were

quantified using HPLC and Elisa kits, respectively. An undisturbed control (CTRL) group was

included for most experimental comparisons. Chronic CORT strongly increased 5-HT7R-LI in

the outer adrenal cortex, as well as 5-HT7 receptor protein and mRNA in whole adrenal glands;

adrenal 5-HT content also increased in these animals. Decreased ACTH and CORT secretion at

30 min of restraint occurred in CORT-treated rats. Results support the notion that chronic stress-

induced increase of adrenocortical 5-HT7 receptors and adrenal 5-HT content is a glucocorticoid-

dependent phenomenon; the development of magnified stress-induced 5-HT7 receptor-mediated

CORT responses in chronically stressed animals nevertheless likely involves additional

mechanisms.

Key words: ACTH; adrenal cortex; chronic corticosterone treatment; chronic stress; 5-HT7

receptors

Page 2 of 30



Draft

3

Introduction

Chronic stress-induced dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis

leading to increased secretion of glucocorticoid hormones (cortisol in humans and corticosterone

[CORT] in rodents) is believed to play a key role in the pathogenesis of a number of stress-

related diseases, including mood disorders such as major depression and anxiety, and a number of

systemic and metabolic pathologies, including arterial hypertension, metabolic syndrome and

type 2 diabetes, among others (Charmandari et al. 2005; Chrousos 2000; Chrousos and Gold

1992; Chrousos 2009). The fundamental mechanisms involved in chronic stress-induced

dysfunction of the HPA axis however have not been elucidated thus far.

Accumulating evidence suggests that ACTH-independent corticosteroid-producing pathways

may be ectopically expressed in the adrenal cortex under conditions of high circulating levels of

glucocorticoids. In this regard, our group recently reported that 14-day chronic exposure to

restraint stress in rats increases restraint-induced CORT secretion through a 5-HT7 receptor-

mediated mechanism, which seems to be ACTH-independent and involves remarkable expression

of 5-HT7 receptors in the adrenal cortex as well as increased adrenal levels of serotonin (5-

hydroxytryptamine; 5-HT) (García-Iglesias et al. 2013). These observations are in line with

clinical pathophysiological studies showing a relationship between states of high circulating

levels of glucocorticoids (e.g. ACTH-independent macronodular adrenal hyperplasias [AIMAHs]

causing Cushing syndrome) and abnormal (i.e. ectopic) expression of adrenocortical 5-HT7

receptors (Louiset et al. 2008; Louiset et al. 2006). To further explore the possible relationship

between increased glucocorticoid levels and expression of adrenocortical 5-HT7 receptors as a

potential pathophysiological mechanism underlying magnified stress-induced corticosteroid

secretion, the present study analysed the effect of a 14-day chronic CORT treatment on

Page 3 of 30



Draft

4

expression and distribution of 5-HT7 receptors in the adrenal glands, adrenal 5-HT content and

baseline and acute restraint-induced ACTH and CORT secretion in rats.

Material and methods

Animals

Male Wistar rats (200-220 g of body weight; n=90) from our own Institutional inbred facilities

were used. Animals (five per cage) were kept at constant temperature (22±1ºC) and humidity (50-

55%) under a 12:12 h: light/dark cycle (lights on 06:00-18:00 h) with food and water available ad

libitum. All the procedures and protocols complied with Federal regulations and were performed

in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, National

Academies Press) and approved by the CINVESTAV-IPN ethics committee (Comité Interno para

el Cuidado y Uso de los Animales de Laboratorio; CICUAL). Efforts were made to minimize

unnecessary suffering of the animals and their number.

Animal groups and chronic corticosterone treatment

A 14-day chronic treatment with CORT (20 mg/kg, s.c. per day) or its vehicle (VEH; 20% 2-

-Hydroxypropyl-β-cyclodextrin; 1 mL/kg, s.c. per day) was given to the animals (between 8:00

and 12:00 h). In order to reduce stress, both VEH and CORT were administered subcutaneously

at the nape of the neck with the animals being slightly restrained. A control (CTRL) home cage

animal group was included for most experimental comparisons.

Determination of body, adrenal gland and thymus weight

Body weight was recorded before and after VEH and CORT treatments. One day after

completion of the treatments (i.e. on day 15), animals were euthanized and adrenal glands from

Page 4 of 30



Draft

5

both sides and thymus were removed and weighed. To exclude possible differences in the weight

of adrenal glands and thymus derived from normal changes in body weight, particularly in

animals receiving CORT treatment in which these parameters are reportedly altered (Coburn-

Litvak et al. 2003; Kaplowitz et al. 2016) organ weights were expressed in milligrams per 100 g

of the final body weight. These somatometric parameters were also recorded in a CTRL animal

group.

Immunohistochemistry assays

One day after VEH and CORT treatments (i.e. on day 15) animals of each group (n=6) were

anesthetized with sodium pentobarbital (60-70 mg/kg, i.p.) and perfused via the ascending aorta

with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 0.1 M phosphate-buffered 4%

paraformaldehyde (pH 7.4). A CTRL animal group (n=6) was included for most comparisons.

The adrenal glands were removed, post-fixed for 24 h at 4ºC, and stored in PBS containing

30% sucrose at 4ºC until use; only the left adrenals were used for the assays. Adrenal sections (35

μm-thick) were obtained using a freezing microtome and collected in culture wells containing

PBS and stored at 4ºC overnight. On the day of the assay, sections were incubated in PBS for 10

min, and then washed four times with PBS containing 0.3% Triton X-100. For antigen retrieval,

sections were incubated in citrate buffer for 10 sec in microwave, washed three times with PBS

containing 0.3% Triton X-100 and then incubated in PBS containing 3% hydrogen peroxide for

30 min. After three more washes with PBS, sections were incubated with 0.3% bovine serum

albumin (BSA) in PBS containing 0.1% Triton X-100 for 2 h at room temperature. Next, sections

were incubated with a primary antibody raised in goat against 5-HT7 receptors (1:200; Santa

Cruz Biotechnology Inc., cat. SC-19160, Santa Cruz, CA, USA) for 70 h at 4ºC in PBS

containing 0.1% Triton X- 100. After three washes with PBS, sections were incubated with a

Page 5 of 30



Draft

6

HRP-conjugated rabbit anti-goat secondary antibody (1:1000; ZyMax, Invitrogen, cat. 81-1620,

Camarillo, CA, USA) for 2 h at room temperature in PBS. After three washes in PBS, sections

were revealed with 3,3′-diaminobenzidine (DAB substrate Kit; Vector Laboratories, Burlingame,

CA, USA) in PBS. Sections were washed with distilled water, mounted on gelatin-coated slides,

cover-slipped, allowed to dry overnight and photographed with a digital camera.

Western blot analysis

One day after completion of CORT and VEH treatments, animals of each group (n=4) were

euthanized by decapitation, and adrenal glands were removed and frozen with liquid nitrogen; a

CTRL animal group (n=4) was included for comparison. Left adrenals were then homogenized in

0.1 M Tris buffer (pH 7.4) and 25X complete protease-free inhibitor cocktail (complete mini

EDTA-free; Roche Applied Science, Penzberg, Germany), and 0.2 M sodium orthovanadate (pH

10; Sigma-Aldrich Inc., St. Louis, MO, USA) using a Polytron (Kinematica AG, Luzern,

Switzerland). Adrenal gland samples from each group were pulled together and the assays were

performed in quadruplicate. The adrenal gland homogenates were sonicated and centrifuged at

13,000 rpm for 15 min at 4°C and the supernatants were collected. Samples of the frontal cortex

were employed as a positive control expressing the 5-HT7 receptor (García-Osta et al. 2000).

Protein determinations were carried out using the Lowry method (Lowry et al. 1951). Identical

amounts of protein (50 μg per lane) of adrenal gland samples were separated by 10% sodium

dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Next, the gels were

transferred on to polyvinylidene difluoride membranes (PVDF; Immobilon transfer membranes,

Millipore Corporation, Billerica, USA). PVDF membranes were then blocked with 5% non-fat

milk in Tris buffered saline-0.1% Tween-20 (TBST buffer, pH 7.4) and then incubated with a

primary antibody raised in goat against the 5-HT7 receptor (1:200; Santa Cruz Biotechnology

Page 6 of 30



Draft

7

Inc., cat. SC-19160, Santa Cruz, CA, USA), or with a primary antibody raised in rabbit against

the control protein, GAPDH (1: 25,000; GeneTex, cat. GTX100118, Alton Pkwy Irvine, CA,

USA), in 5% non-fat milk with TBST for overnight at 4°C. Next, membranes were rinsed three

times with TBST and then incubated with a rabbit anti-goat HRP-conjugated secondary antibody

(1:1000; ZyMax, Invitrogen, cat. 81-1620, Camarillo, CA, USA) or with a goat anti-rabbit HRP-

conjugated secondary antibody (1:5000; Santa Cruz Biotechnology Inc. cat. Sc 2004, Heidelberg

Germany) in 5% non-fat milk with TBST for 2 h, respectively. Membranes were then rinsed three

times with TBST and developed afterwards by incubation with chemiluminescent reagent

(SuperSignal West Femto Maximum Sensitivity Substrate, Thermo scientific, Rockford, USA).

To estimate the amount of 5-HT7 receptor protein relative to GAPDH for each sample, a

densitometric analysis was performed using the Quantity One® Software (Bio-Rad).

Reverse transcription-polymerase chain reaction

The adrenal glands from VEH- and CORT-treated animals (n=4 each) were excised and those of

the left side were used for the assays; a home cage CTRL group (n=4) was also included for

comparison. Expression of the mRNA encoding for the 5-HT7 receptor was measured by reverse

transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted using the TRIzol

reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was conducted in a reaction

volume of 50 µL using 5 µg of total RNA and Super Script II One-Step RT-PCR system

(Invitrogen, Carlsbad, CA, USA), following the manufacturer´s protocol in an end-point thermal

cycler (Gene Cycler; Bio-Rad). For amplification of 5-HT7 receptor cDNA, primers for rat 5-

HT7 receptor sequences were: sense 5’-AGG ATT TTG GCT ACA CGA TC-3’, and antisense,

5’-GAG GAA AAA CGG CAG CCA GCA-3’. These primer sequences have been used in

previous studies reporting expression of the 5-HT7 receptor in rat and porcine tissues (Turner et

Page 7 of 30



Draft

8

al. 2003; Ullmer et al. 1995; Urbina et al. 2014). Negative controls were made with total RNA to

ensure avoidance of genomic contamination. Positive and negative controls for 5-HT7 receptor

expression were carried out with total RNA from rat frontal cortex (García-Osta et al. 2000) and

liver (Shen et al. 1993), respectively. The constitutive GAPDH gene was used as housekeeping

gene with the primers: sense 5’-ACC ACA GTC CAT GCC ATC AG-3’, and antisense 5’-TCC

ACC ACC CTG TTG CTG TA-3’. The amplification profile involved a cDNA synthesis cycle at

60oC for 30 min, a denaturation cycle at 94ºC for 2 min, and 35 cycles involving denaturation at

92ºC for 1 min, annealing at 60ºC for 1 min, and extension at 74ºC for 2 min. After amplification,

PCR products were electrophoresed on a 1.5 % agarose gel for 1 h at 94 volts. Subsequent to

agarose gel electrophoresis bands were visualized with ethidium bromide under a UV light lamp

and digitalized; then, their intensities were measured by densitometry using the Quantity One 1-D

Image Analysis Software (Bio-Rad Laboratories Inc.). Since GAPDH mRNA expression did not

vary among treatment groups, all samples were normalized against this.

High Performance Liquid Chromatography

In order to verify whether chronic CORT treatment would also increase the adrenal content of 5-

HT, as reported in animal with chronic restraint stress (García-Iglesias et al. 2013), baseline

levels of 5-HT in adrenal glands from CTRL, VEH- and CORT-treated animals (n=4 each) were

determined by reverse-phase HPLC with electrochemical detection, as previously described for

dopamine (Gonzalez-Barrios et al. 2006). Briefly, one day after treatments (i.e. on day 15), left

adrenal glands were obtained from decapitated animals and thoroughly washed in isotonic saline

to remove remaining blood. The adrenals were then individually homogenized in cold 0.1 M

perchloric acid and ultracentrifuged at 10 psi for 10 min; supernatants were collected and filtered

(0.22 mm nylon filters; Millipore). Samples were then separated with an ODS Velosep RP C18

Page 8 of 30



Draft

9

column (3 µm, 100 x 3.2 mm, PerkinElmer, Waltham, MA, USA) at 30.5 °C, and detected by a

LC-4C electrochemical detector at +0.75 V with respect to the Ag/AgCl reference electrode

(Bioanalytical Systems, West Lafayette, IN, USA). Next, samples were eluted with a mobile

phase containing 0.01 M sodium dihydrogen phosphate, 0.01 M citric acid, 2 mM sodium EDTA,

1 mM octanesulfonic acid/sodium salt and 10% methanol at pH 3.22; the mobile phase was

delivered by a BAS HPLC PM-80- Pump (Bioanalytical Systems Inc., West Lafayette, IN, USA)

in isocratic elution mode at 0.5 mL/min. Quantification of 5-HT was performed with the Chrom

Graph 2.34.00 REPORT 2.30 software (Bioanalytical Systems, Inc., West Lafayette, IN, USA)

and protein concentrations were determined by the Bradford method (Bradford 1976).

Concentrations of 5-HT were calculated from the area of the chromatographic peaks based on an

internal standard and expressed as ρg/µg of protein.

Neuroendocrine studies

One day after the end of VEH and CORT treatments (i.e. on day 15), animals from each group

were subdivided into three subgroups, which were either left undisturbed (0 min; n=6) or

submitted to an acute restraint session for 10 (n=6) and 30 min (n=6), respectively. After the

acute stress periods, animals were killed by decapitation and trunk blood samples collected and

centrifuged; animals with no acute stress (i.e. 0 min) were decapitated before the beginning of

restraint sessions. Plasma was stored at -80ºC until used. Commercially available Elisa kits for

ACTH (cat. EK-001-21, Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) and CORT (cat.

ADI-900-097, Enzo, Farmingdale, NY, USA) were employed for hormone level measurements.

The minimum detection concentration for each assay was 0.1 ng/mL and 27 pg/mL, respectively,

with an intra-assay precision of

Draft

10

Data presentation and statistical evaluation

All data in the text, table and figures are presented as the mean ± SEM of at least four

determinations. The differences in body weight gain and relative organ weight, relative tissue

content of 5-HT7 receptor protein and mRNA as well as adrenal 5-HT content between CTRL,

VEH- and CORT-treated animals were all compared by one-way ANOVA. The effects of chronic

VEH and CORT treatments on acute restraint-induced ACTH and CORT secretion were

compared by a two-way ANOVA, with time of restraint and chronic treatment as between-

subject independent factors. The ANOVA tests were followed by Bonferroni post-hoc tests to

determine differences. In all cases, the level of significance was set at P < 0.05. Statistical

analyses were performed using GraphPad Prism version 6.00 (GraphPad Software, La Jolla,

California, USA).

Results

Effect of treatments on somatometric parameters

The physiological parameters reportedly altered subsequent to chronic stress exposure were

determined. Animals that received chronic CORT treatment exhibited significantly lower total

body weight numbers, which was reflected as a 15.4% and 22.7% loss of body weight gain as

compared to VEH-treated and CTRL animals, respectively (Table 1). In contrast to previous

observations showing adrenal gland enlargement in chronically-stressed animals (García-Iglesias

et al. 2013; Ulrich-Lai et al. 2006), relative adrenal gland weight was significantly lower in

CORT- as compared to VEH-treated and CTRL animals (Table 1). In the case of relative thymus

weight a decrease was observed in animals that received CORT relative to CTRL and VEH

treatments (Table 1).

Page 10 of 30



Draft

11

Effect of chronic corticosterone treatment on 5-HT7 receptor expression in adrenal glands

The impact of chronic corticosteroid treatment on 5-HT7 receptor expression in the adrenal

glands was evaluated by immunohistochemistry, Western blot and RT-PCR assays. Thus, in

resemblance to previously reported changes in the amount of 5-HT7 receptors in the adrenal

glands from animals with a history of 14-day chronic restraint stress (García-Iglesias et al. 2013),

chronic administration of CORT induced, relative to CTRL conditions (Fig. 1A, 1D) and VEH

treatment (Fig. 1B, 1E), a remarkable increase of 5-HT7 receptor-like immunoreactivity (5-

HT7R-LI) in the adrenal cortex, with the highest density of immunostaining in the zona

glomerulosa and the outer zona fasciculata cell layers (Fig. 1C, 1F). In addition, higher 5-HT7R-

LI was detected in the cortex of adrenal glands from VEH-treated (Fig. 1B, 1E) as compared to

CTRL animals (Fig. 1A, 1D) most likely as a result of stress induced by daily manipulation and

injection in the former group.

Consistent with the above observations, Western blot analyses revealed a 56.9% and 57.4%

higher relative 5-HT7 receptor protein content in adrenal glands from CORT-treated rats as

compared to the corresponding values in adrenals from VEH-treated and CTRL animals (Fig.

2A). That the above changes in protein content may be accounted for by increased expression of

5-HT7 receptors is supported by the observation that the corresponding mRNA levels also

increased in the adrenal glands from CORT-treated relative to CTRL and VEH-treated animals as

shown by the RT-PCR experiments (Fig. 2B). Interestingly, these experiments revealed the

presence of two distinct bands (i.e. adrenals from CORT-treated animals and frontal cortex; Fig.

2B) likely suggesting the expression of two 5-HT7 receptor isoforms in these tissues.

Page 11 of 30



Draft

12

Effect of chronic corticosterone treatment on adrenal 5-HT content

As shown in Fig. 3, chronic CORT administration induced a significant increase in the adrenal

content of 5-HT as compared to that in adrenals from CTRL and VEH-treated animals. As one

could expect from the stressful effects of manipulation in animals that received chronic VEH

administration, a higher adrenal content of 5-HT was detected in this group as compared to that in

CTRL animals (Fig. 3).

Effect of treatments on acute restraint-induced ACTH and corticosterone secretion

As depicted in Fig. 4, acute restraint induced a time of stress-dependent increase of ACTH and

CORT secretion in VEH-treated rats with the highest secretion of both hormones occurring at the

30 min restraint period. In contrast to previous observations in animals with a history of chronic

restraint stress exhibiting markedly blunted restraint-induced ACTH secretion along with

magnified restraint-induced CORT secretory responses (García-Iglesias et al. 2013), chronic

CORT administration produced a modest non-significant blunting of both baseline (0 min) levels

and stress-induced ACTH and CORT responses at 10 min of restraint, along with a significant

decrease of stress-induced responses of both hormones at 30 min of restraint (Fig. 4).

Discussion

The major finding of the present study is that raising the circulating levels of CORT through a

14-day chronic administration protocol did increase expression of 5-HT7 receptors in the adrenal

cortex relative to corresponding tissue samples from CTRL and VEH-treated animals. This effect

was visualized as a remarkable increase of 5-HT7R-LI in adrenocortical cells of the zona

glomerulosa and the outer and inner zona fasciculata, along with increased 5-HT7 receptor

protein and mRNA levels in whole adrenal glands from animals that received CORT as compared

Page 12 of 30



Draft

13

to those that received VEH or no treatment at all (CTRL group). In support of a role of

glucocorticoids in the development of a stimulatory serotonergic loop in the adrenal glands as a

result of chronic stress exposure (García-Iglesias et al. 2013), chronic CORT treatment also

increased the adrenal content of 5-HT as compared to CTRL and VEH treatments. Apart from the

implications discussed below, the present results support the notion that ectopic expression of

adrenocortical 5-HT7 receptors (likely involved in stress-induced corticosteroid secretion) may

be an ACTH-dissociated glucocorticoid-dependent phenomenon. This CORT-induced change

however did not correlate with sensitized restraint-induced CORT secretory responses.

The typical morphometric changes induced by chronic stress include decrease of body

weight gain, thymus involution and adrenal enlargement (Blanchard et al. 1998; Gamallo et al.

1986; Hu et al. 2000; Kuipers et al. 2003; Moraska et al. 2000). In the present study, chronic

corticosteroid treatment also decreased body weight gain and relative thymus weight but it

produced however a decrease of relative adrenal weight. This later finding, which parallels

previous observations showing a decrease of adrenal mass in rats with chronically elevated

CORT levels (D’souza et al. 2012), suggests that it is the chronic stimulation of the stress

response but not the sole increase of glucocorticoid levels what may actually induce enlargement

of the adrenal glands. It is well known in fact that during prolonged ACTH treatment an initial

increase in adrenal weight, RNA and protein, which is later followed by an increase in adrenal

DNA takes place (Alario et al. 1987; Dallman et al. 1980; Imrie et al. 1965), consistent with the

notion that stress-induced surges of ACTH play a critical role in chronic stress-induced adrenal

enlargement through mechanisms that involve hyperplasia and hypertrophy of the outer and inner

zona fasciculata cell layers, respectively (Ulrich-Lai et al. 2006). Accordingly, chronic ACTH

treatment reportedly increases the volume of zona fasciculata cells (Nussdorfer and Mazzocchi

1983), which in turn correlates with increased corticosteroid output in response to exogenous

Page 13 of 30



Draft

14

ACTH without detectable changes in adrenal ACTH sensitivity (Ulrich-Lai et al. 2006). Then,

the decrease of relative adrenal weight in CORT- as compared to CTRL and VEH-treated

animals (present study) was most likely due to the absence of ACTH secretory responses.

Accumulating evidence suggests that adrenocortical 5-HT7 receptors may play an important

pathophysiological role in states of hypercortisolemia (Brooks et al. 1988; García-Iglesias et al.

2013; Louiset et al. 2006; Louiset et al. 2008) and that selective 5-HT7 receptor antagonists

might in fact represent a new therapeutic approach for the treatment of stress-related disorders

including major depression (Hedlund et al. 2005; Wesołowska et al. 2006a; Wesołowska et al.

2006b; Wesołowska et al. 2007). A role of 5-HT7 receptors in chronic stress-induced endocrine

dysregulation and excessive stress-induced corticosteroid secretion has been proposed on the

basis of: a) the effectiveness of a selective 5-HT7 receptor antagonist to restore sensitized

restraint-induced CORT responses to control values in animals with a previous experience of

chronic stress (García-Iglesias et al. 2013); b) ectopic expression of adrenocortical 5-HT7

receptors under conditions of high circulating levels of glucocorticoids, as reported in chronically

stressed animals (García-Iglesias et al. 2013) as well as in AIMAH (Louiset et al. 2006) and

carcinoma cells ( Louiset et al. 2008); interestingly, 5-HT has been found to produce more potent

and efficacious corticosteroid secretory responses in adrenocortical adenoma cells in vitro (as

compared to normal tissue cells) through a methiothepin-sensitive mechanism (Contesse et al.

2005); and c) increased 5-HT-like immunoreactivity in the adrenal cortex and 5-HT content in

adrenals from animals previously submitted to chronic stress (García-Iglesias et al. 2013). In the

present study we found that raising the circulating levels of corticosteroids through a 14-day

chronic CORT treatment did induce expression of 5-HT7 receptors as shown by the increase of

adrenal 5-HT7 receptor protein and mRNA levels. These observations are in resemblance with

earlier reports showing that acute stress increases 5-HT7 receptor mRNA in the rat hippocampus

Page 14 of 30



Draft

15

(Yau et al. 2001), and that chronic unpredictable mild stress enhances it both in the hippocampus

and the hypothalamus (Li et al. 2009). In contrast to these observations however, blunting of

glucocorticoid levels by adrenalectomy was shown to induce 5-HT7 receptor upregulation in the

rat hippocampus (Yau et al. 1997) whereas chronic restraint stress decreased 5-HT7 receptor

levels in the paraventricular nucleus of the hypothalamus in rats (García-Iglesias et al. 2013).

Together, the above observations reveal complex and contrasting effects of glucocorticoids on

regulation of 5-HT7 receptor expression, the nature of which remains to be elucidated. Finally,

regarding the present RT-PCR results, this is the first study showing a potential expression of 5-

HT7 receptor isoforms in rat adrenal glands. Interestingly, a previous study using the same

primer sequences employed here (Ullmer et al. 1995) reported a closely similar pattern of 5-HT7

receptor mRNA expression in porcine conjunctival cells (i.e. two distinct bands; Turner et al.

2003). Further studies are required to explore the possible expression of 5-HT7 receptor isoforms

in rat adrenal glands and whether this might be affected by chronic exposure to glucocorticoids.

In addition to increasing 5-HT7 receptor expression, chronic CORT administration also

produced an increase in the adrenal content of 5-HT. This finding supports the notion that the

increased adrenal levels of 5-HT in animals with a history of chronic restraint stress (García-

Iglesias et al. 2013) is also a glucocorticoid-dependent phenomenon; thus, 5-HT would represent

a stimulatory signal capable of activating corticosteroid secretion via activation of adrenocortical

5-HT7 receptors. It remains to be determined nevertheless whether the above effect of

glucocorticoids on adrenal 5-HT levels may involve synthesis of this monoamine, as previously

observed in primary pigmented nodular adrenocortical disease (i.e. a clinical pathological

condition of ACTH-independent hypercortisolemia; Bram et al. 2016).

The dose of CORT employed in the present study (20 mg/kg, s.c. per day) was selected on

the basis of previous studies showing that a chronic 7-day CORT treatment given in a total dose

Page 15 of 30



Draft

16

of 20 mg/kg per day (10 mg/kg, s.c. given twice a day) induced both functional desensitization of

5-HT1A receptors in the CA1 area of the hippocampus (Czyrak et al. 2002) and regulation of

function and expression of 5-HT1A receptors involved in disruption of prepulse inhibition of

acute startle response in rats (Czyrak et al. 2003). Further, it should be highlighted that, while

preparing this manuscript, an interesting study appeared published on the ability of a 14-day

chronic CORT treatment (10 mg/kg, s.c. given twice a day) to reduce the reactivity of 5-HT7

receptors that modulate GABAergic transmission in the dorsal raphe nucleus, thus supporting the

ability of our chronic CORT treatment protocol to induce significant alterations of the 5-HT

system and receptors (Sowa et al. 2018).

From the results of the immunohistochemistry assays, it seems clear that 5-HT7 receptor

expression in CORT-treated rats occurred primarily in the adrenal cortex but not in the medulla;

such adrenocortical changes were observed in the zona glomerulosa and the outer and inner zona

fasciculata cell layers thereby resembling the expression pattern reported in animals with chronic

restraint stress (García-Iglesias et al. 2013). Indeed, previous PCR and Western blot experiments

have revealed expression of 5-HT7 receptor mRNA and protein in the zona fasciculata/reticulata

of the rat adrenal cortex (Contesse et al. 1999), which is in line with the present

immunohistochemistry and Western blot results. It should be highlighted nevertheless that

expression of 5-HT7 receptors in adrenocortical corticosteroid-producing cells did not translate

into augmented restraint-induced CORT responses, as reported previously in rats with a history

of a 14-day chronic stress paradigm (García-Iglesias et al. 2013). This implies that more complex

mechanisms must be set in motion (e.g. by stress) for the development and manifestation of

corticosteroid hypersecretion. A likely explanation for the contrasting effects between chronic

stress and chronic CORT treatment regarding acute stress-induced corticosteroid secretion may

rely on the absence of repeated ACTH secretory responses in the latter (present study). In

Page 16 of 30



Draft

17

accordance with this possibility, previous in vitro studies using dispersed zona fasciculata cells

have demonstrated that ACTH evokes grater maximal cAMP, pregnenolone and CORT

production in cells taken from rats with a previous experience of chronic restraint stress (2 h/day

for 14 days) with no change in sensitivity; interestingly, these alterations were mimicked by

repeated ACTH administration thus implicating an ACTH-dependent mechanism (Aguilera et al.

1996). Similar findings were reported in dexamethasone-blocked rats in which a 14-day chronic

variable stress paradigm produced increased CORT responses to exogenous ACTH

administration with no change in sensitivity (Ulrich-Lai et al. 2006). Together, these observations

suggest that, upon exposure to chronic stress, ACTH responses may promote increased ACTH

receptor expression and/or activity leading in turn to magnified activity of corticosteroid-

producing pathways. This notion is consistent with the well-documented effects of ACTH in

adrenal steroidogenesis, which comprise important regulatory effects on gene transcription of

steroidogenic enzymes in the adrenal cortex (Davis and Garren 1968; Pon and Orme-Johnson

1984). Accordingly, repeated ACTH administration reportedly increases the levels of

steroidogenic acute regulatory protein (StAR) mRNA and protein in the zona fasciculata-

reticularis (Lehoux et al. 1998), whereas a 3-fold increase of ACTH plasma levels through

metyrapone administration in rats was found to increase zona fasciculata cells containing the

glucocorticoid synthesizing enzyme, cytochrome P45011β (Mitani et al. 1996). On the basis of

the above, the sensitizing effects of chronic stress on acute stress-induced corticosteroid secretion

may require, at least in part, ACTH-dependent sensitization of the signalling pathways involved

in corticosteroid production. Nevertheless, in the light of the remarkable blunting of stress-

induced ACTH responses in animals with a history of chronic restraint stress (García-Iglesias et

al. 2013), a key issue to be clarified is whether additional sensitizing mechanisms of

corticosteroid production may come into play during the course of chronic stress once

Page 17 of 30



Draft

18

desensitization of the ACTH output to the persistent stimulus has taken place, as reported in

studies with other physical/psychological stress paradigms (see Aguilera 1994 for review).

Finally, regarding the molecular mechanisms potentially involved in chronic stress-induced

5-HT7 receptor overexpression and 5-HT-producing pathways in adrenocortical cells (García-

Iglesias et al. 2013), previous studies in human adrenocortical cells have shown that activation of

protein kinase A (PKA) by genetic mutations affecting the PKA regulatory subunit R1A, triggers

upregulation of the 5-HT synthesizing enzyme, tryptophan hydroxylase, and several types of 5-

HT receptors including the 5-HT7 receptor, leading to enhancement of glucocorticoid production

(Bram et al. 2016). Since ACTH is well known to trigger glucocorticoid secretion through

activation of the cAMP/PKA pathway after binding to its specific receptor (i.e. the MC2

receptor), it seems likely that ACTH may initiate 5-HT7 receptor overexpression and 5-HT

synthesis in the adrenal glands during chronic stress, the synthesis of both being then amplified

by CORT produced under ACTH stimulation. On this basis, 5-HT7 receptor expression and

probably also 5-HT synthesis could thus result from a dual mechanism that would synergistically

or sequentially involve both ACTH and CORT. Further investigation aimed at elucidating

detailed molecular mechanisms involved in chronic stress-induced hypercortisolemia is

warranted.

Conclusion

The results of the present study are consistent with the notion that ectopic expression of 5-HT7

receptors and 5-HT levels in adrenocortical steroidogenic cells is a glucocorticoid-dependent

phenomenon. Relative to previous observations in rats with a history of chronic restraint stress

however, the absence of sensitized restraint-induced corticosteroid responses in CORT-treated

animals implies that, further to adrenocortical 5-HT7 receptors, other mediators/mechanisms

Page 18 of 30



Draft

19

must be involved in chronic stress-induced endocrine dysregulation and increased corticosteroid

secretion. Further investigation is required to elucidate the molecular mechanisms underlying

abnormal ACTH-independent secretion of glucocorticoids in which adrenocortical 5-HT7

receptors seem to play a role. It also remains to be determined whether blockade of

adrenocortical 5-HT7 receptors involved in hypercortisolemia might account for the

antidepressant-like effects of 5-HT7 receptor antagonist drugs (Hedlund et al. 2005; Wesołowska

et al. 2006a; Wesołowska et al. 2006b; Wesołowska et al. 2007).

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

Authors are grateful to Dr. Daniel Martínez-Fong (Department of Physiology, Biophysics and

Neurosciences, CINVESTAV-IPN, Mexico City), for providing us with guidance and technical

facilities for HPLC measurements. This study was supported by the National Research Council of

Science and Technology of Mexico (CONACYT; Grant 256882).

References

Aguilera G. 1994. Regulation of pituitary ACTH secretion during chronic stress. Frontiers in Neuroendocrinology, 15(4): 321–350. https://doi.org/10.1006/frne.1994.1013

Aguilera G., Kiss A., Lu A., and Camacho C. 1996. Regulation of adrenal steroidogenesis during chronic stress. Endocr Res. 22(4): 433–443.

Alario P., Gamallo A., Beato M. J., and Trancho G. 1987. Body weight gain, food intake and adrenal development in chronic noise stressed rats. Physiol Behav. 40(1): 29–32.

Blanchard R. J., Nikulina J. N., Sakai R. R., McKittrick C., McEwen B., and Blanchard D. C. 1998. Behavioral and endocrine change following chronic predatory stress. Physiol Behav. 63(4): 561–569.

Bradford M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72: 248–254.

Page 19 of 30



Draft

20

Bram Z., Louiset E., Ragazzon B., Renouf S., Wils J., Duparc C., et al. 2016. PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease. JCI Insight, 1(15): e87958. https://doi.org/10.1172/jci.insight.87958

Brooks B. A., McBride O. W., Dolphin C. T., Farrall M., Scambler P. J., Gonzalez F. J., and Idle J. R. 1988. The gene CYP3 encoding P450pcn1 (nifedipine oxidase) is tightly linked to the gene COL1A2 encoding collagen type 1 alpha on 7q21-q22.1. Am J Hum Genet. 43(3): 280–284.

Charmandari E., Tsigos C., and Chrousos G. 2005. Endocrinology of the stress response. Annu Rev Physiol. 67: 259–284. https://doi.org/10.1146/annurev.physiol.67.040403.120816

Chrousos G. P. 2000. The stress response and immune function: clinical implications. The 1999 Novera H. Spector Lecture. Ann N Y Acad Sci. 917: 38–67.

Chrousos G. P. and Gold P. W. 1992. The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA, 267(9): 1244–1252.

Chrousos G. P. 2009. Stress and disorders of the stress system. Nat Rev Endocrinol. 5(7): 374–381. https://doi.org/10.1038/nrendo.2009.106

Coburn-Litvak P. S., Pothakos K., Tata D. A., McCloskey D. P., and Anderson B. J. 2003. Chronic administration of corticosterone impairs spatial reference memory before spatial working memory in rats. Neurobiol Learn Mem. 80(1): 11–23.

Contesse V., Lenglet S., Grumolato L., Anouar Y., Lihrmann I., Lefebvre H., et al. 1999. Pharmacological and molecular characterization of 5-hydroxytryptamine(7) receptors in the rat adrenal gland. Mol Pharmacol. 56(3): 552–561.

Contesse V., Reznik Y., Louiset E., Duparc C., Cartier D., Sicard F., et al. 2005. Abnormal sensitivity of cortisol-producing adrenocortical adenomas to serotonin: in vivo and in vitro studies. J Clin Endocrinol Metab. 90(5): 2843–2850. https://doi.org/10.1210/jc.2004-2476

Czyrak A., Maćkowiak M., Chocyk A., Fijał K., Gadek-Michalska A., and Wedzony K. 2003. 8-OHDPAT-induced disruption of prepulse inhibition in rats is attenuated by prolonged corticosterone treatment. Neuropsychopharmacology, 28(7): 1300–1310. https://doi.org/10.1038/sj.npp.1300165

Czyrak A., Maćkowiak M., Chocyk A., Fijał K., Tokarski K., Bijak M., and Wedzony K. 2002. Prolonged corticosterone treatment alters the responsiveness of 5-HT1A receptors to 8-OH-DPAT in rat CA1 hippocampal neurons. Naunyn Schmiedebergs Arch Pharmacol. 366(4): 357–367. https://doi.org/10.1007/s00210-002-0586-2

Dallman M. F., Engeland W. C., Holzwarth M. A., and Scholz P. M. 1980. Adrenocorticotropin inhibits compensatory adrenal growth after unilateral adrenalectomy. Endocrinology, 107(5): 1397–1404. https://doi.org/10.1210/endo-107-5-1397

Davis W. W., and Garren L. D. 1968. On the mechanism of action of adrenocorticotropic hormone. The inhibitory site of cycloheximide in the pathway of steroid biosynthesis. J Biol Chem. 243(19): 5153–5157.

D’souza A. M., Beaudry J. L., Szigiato A. A., Trumble S. J., Snook L. A., Bonen, A., et al. 2012. Consumption of a high-fat diet rapidly exacerbates the development of fatty liver disease that occurs with chronically elevated glucocorticoids. Am J Physiol Gastrointest Liver Physiol. 302(8): G850-863. https://doi.org/10.1152/ajpgi.00378.2011

Gamallo A., Villanua A., Trancho G., and Fraile A. 1986. Stress adaptation and adrenal activity in isolated and crowded rats. Physiol Behav. 36(2): 217–221.

Page 20 of 30



Draft

21

García-Iglesias B. B., Mendoza-Garrido M. E., Gutiérrez-Ospina G., Rangel-Barajas C., Noyola-Díaz M., and Terrón J. A. 2013. Sensitization of restraint-induced corticosterone secretion after chronic restraint in rats: involvement of 5-HT₇ receptors. Neuropharmacology, 71: 216–227. https://doi.org/10.1016/j.neuropharm.2013.03.013

García-Osta A., Frechilla D., and Del Río J. 2000. Effect of p-chloroamphetamine on 5-HT1A and 5-HT7 serotonin receptor expression in rat brain. J Neurochem, 74(5): 1790–1797.

Gonzalez-Barrios J. A., Lindahl M., Bannon M. J., Anaya-Martínez V., Flores G., Navarro-Quiroga I., et al. 2006. Neurotensin polyplex as an efficient carrier for delivering the human GDNF gene into nigral dopamine neurons of hemiparkinsonian rats. Mol Ther, 14(6), 857–865. https://doi.org/10.1016/j.ymthe.2006.09.001

Hedlund P. B., Huitron-Resendiz S., Henriksen S. J., and Sutcliffe J. G. 2005. 5-HT7 receptor inhibition and inactivation induce antidepressantlike behavior and sleep pattern. Biol Psychiatry, 58(10): 831–837. https://doi.org/10.1016/j.biopsych.2005.05.012

Hu Y., Gursoy E., Cardounel A., and Kalimi M. 2000. Biological effects of single and repeated swimming stress in male rats: beneficial effects of glucocorticoids. Endocrine, 13(1): 123–129. https://doi.org/10.1385/ENDO:13:1:123

Imrie R. C., Ramaiah T. R., Antoni F., and Hutchison W. C. 1965. THE EFFECT OF ADRENOCORTICOTROPHIN ON THE NUCLEIC ACID METABOLISM OF THE RAT ADRENAL GLAND. J Endocrinol. 32: 303–312.

Kaplowitz E. T., Savenkova M., Karatsoreos I. N., and Romeo R. D. 2016. Somatic and Neuroendocrine Changes in Response to Chronic Corticosterone Exposure During Adolescence in Male and Female Rats. J Neuroendocrinol. 28(2): 12336. https://doi.org/10.1111/jne.12336

Kuipers S. D., Trentani A., Den Boer J. A., and Ter Horst G. J. 2003. Molecular correlates of impaired prefrontal plasticity in response to chronic stress. J Neurochem. 85(5): 1312–1323.

Lehoux J. G., Fleury A., and Ducharme L. 1998. The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo. Endocrinology, 139(9): 3913–3922. https://doi.org/10.1210/endo.139.9.6196

Li Y. C., Wang F. M., Pan Y., Qiang L. Q., Cheng G., Zhang W. Y., and Kong L. D. 2009. Antidepressant-like effects of curcumin on serotonergic receptor-coupled AC-cAMP pathway in chronic unpredictable mild stress of rats. Prog Neuropsychopharmacol Biol Psychiatry, 33(3): 435–449. https://doi.org/10.1016/j.pnpbp.2009.01.006

Louiset E., Isvi K., Gasc J. M., Duparc C., Cauliez B., Laquerrière A., et al. 2008. Ectopic expression of serotonin7 receptors in an adrenocortical carcinoma co-secreting renin and cortisol. Endocr Relat Cancer, 15(4): 1025–1034. https://doi.org/10.1677/ERC-08-0085

Louiset E., Contesse V., Groussin L., Cartier D., Duparc C., Barrande G., et al. 2006. Expression of serotonin7 receptor and coupling of ectopic receptors to protein kinase A and ionic currents in adrenocorticotropin-independent macronodular adrenal hyperplasia causing Cushing’s syndrome. J Clin Endocrinol Metab. 91(11): 4578–4586. https://doi.org/10.1210/jc.2006-0538

Lowry O. H., Rosebrough N. J., Farr, A. L., and Randall R. J. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1): 265–275.

Mitani F., Miyamoto H., Mukai K., and Ishimura Y. 1996. Effects of long term stimulation of ACTH and angiotensin II-secretions on the rat adrenal cortex. Endocr Res. 22(4): 421–431.

Page 21 of 30



Draft

22

Moraska A., Deak T., Spencer R. L., Roth D., and Fleshner M. 2000. Treadmill running produces both positive and negative physiological adaptations in Sprague-Dawley rats. Am J Physiol Regul Integr Comp Physiol. 279(4): R1321-1329. https://doi.org/10.1152/ajpregu.2000.279.4.R1321

Nussdorfer G. G., and Mazzocchi G. 1983. Long-term effects of ACTH on rat adrenocortical cells: a coupled stereological and enzymological study. J Steroid Biochem. 19(6): 1753–1756.

Pon L. A., and Orme-Johnson N. R. 1984. Protein synthesis requirement for acute ACTH stimulation of adrenal corticosteroidogenesis. Endocr Res. 10(3–4): 585–590.

Shen Y., Monsma F. J., Metcalf M. A., Jose P. A., Hamblin M. W., and Sibley D. R. 1993. Molecular cloning and expression of a 5-hydroxytryptamine7 serotonin receptor subtype. J Biol Chem. 268(24): 18200-18204.

Sowa J., Kusek M., Siwiec M., Sowa J. E., Bobula B., Tokarski K., and Hess G. 2018. The 5-HT7 receptor antagonist SB 269970 ameliorates corticosterone-induced alterations in 5-HT7 receptor-mediated modulation of GABAergic transmission in the rat dorsal raphe nucleus. Psychopharmacology, 235(12): 3381–3390. https://doi.org/10.1007/s00213-018-5045-y

Terrón J. A. 2014. Novel insights into the potential involvement of 5-HT7 receptors in endocrine dysregulation in stress-related disorders. Rev Neurosci. 25(3): 439–449. https://doi.org/10.1515/revneuro-2014-0017

Turner H. C., Alvarez L. J., Candia O. A., and Bernstein A. M. 2003. Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae. Curr Eye Res. 27(4): 205–215.

Ullmer C., Schmuck K., Kalkman H. O., and Lübbert H. 1995. Expression of serotonin receptor mRNAs in blood vessels. FEBS Lett. 370(3): 215–221.

Ulrich-Lai Y. M., Figueiredo H. F., Ostrander M. M., Choi D. C., Engeland W. C., and Herman J. P. 2006. Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner. Am J Physiol Endocrinol Metab. 291(5): E965-973. https://doi.org/10.1152/ajpendo.00070.2006

Urbina M., Arroyo R., and Lima L. 2014. 5-HT7 receptors and tryptophan hydroxylase in lymphocytes of rats: mitogen activation, physical restraint or treatment with reserpine. Neuroimmunomodulation, 21(5): 240–249. https://doi.org/10.1159/000357148

Wesołowska A., Nikiforuk A., and Stachowicz K. 2006. Potential anxiolytic and antidepressant effects of the selective 5-HT7 receptor antagonist SB 269970 after intrahippocampal administration to rats. Eur J Pharmacol. 553(1–3): 185–190. https://doi.org/10.1016/j.ejphar.2006.09.064

Wesołowska A., Nikiforuk A., Stachowicz K., and Tatarczyńska E. 2006. Effect of the selective 5-HT7 receptor antagonist SB 269970 in animal models of anxiety and depression. Neuropharmacology, 51(3): 578–586. https://doi.org/10.1016/j.neuropharm.2006.04.017

Wesołowska A., Tatarczyńska E., Nikiforuk A., and Chojnacka-Wójcik E. 2007. Enhancement of the anti-immobility action of antidepressants by a selective 5-HT7 receptor antagonist in the forced swimming test in mice. Eur J Pharmacol. 555(1): 43–47. https://doi.org/10.1016/j.ejphar.2006.10.001

Yau J. L., Noble J., and Seckl J. R. 2001. Acute restraint stress increases 5-HT7 receptor mRNA expression in the rat hippocampus. Neurosci Lett. 309(3): 141–144.

Page 22 of 30



Draft

23

Yau J. L., Noble J., Widdowson J., and Seckl J. R. 1997. Impact of adrenalectomy on 5-HT6 and 5-HT7 receptor gene expression in the rat hippocampus. Brain Res Mol Brain Res. 45(1): 182–186.

Page 23 of 30



Draft

24

Legends to Figures

Fig. 1. The effect of 14-day chronic administration of corticosterone (CORT; 20 mg/kg, s.c. per

day) as compared to vehicle (VEH; 1 ml/kg, s.c. per day) and home cage control (CTRL)

conditions on 5-HT7 receptor-like immunoreactivity (5-HT7-RLI) in rat adrenal glands. Whereas

5-HT7-RLI was scarcely observed in the cortex of CTRL tissues (A and D), strong 5-HT7

receptor immunostaining was observed in the outer cortical areas of adrenal glands from CORT-

treated animals (C and F). Consistent with the effect of stress induced by daily manipulation and

s.c. administration, increased 5-HT7-RLI was observed in the cortex of adrenals taken from

VEH-treated animals as compared to CTRL tissues (B and E). Bars in A, B and C = 500 µm; bars

in D, E and F = 100 µm



conditions on the content of 5-HT7 receptor protein (panel A) and mRNA (panel B) levels with

respect to GAPDH, as determined by Western blot analysis and RT-PCR, respectively. Chronic

CORT treatment significantly increased both relative 5-HT7 receptor protein and mRNA levels

with respect to CTRL conditions and chronic VEH administration. Samples of the frontal cortex

(FC) and liver (LIV) were taken from CTRL animals and assayed as positive and negative

controls, respectively, of 5-HT7 receptor expression. ** P < 0.01 vs CTRL; *** P < 0.001 vs

CTRL.

Page 24 of 30



Draft

25



conditions on the adrenal content of 5-HT as measured by HPLC. Chronic CORT treatment

significantly increased adrenal 5-HT levels as compared to CTRL and VEH treatments; in

addition, a higher content of 5-HT was detected in VEH-treated animals as compared to CTRL

tissue samples. ** P < 0.01 vs VEH; *** P < 0.001 vs CTRL.

Fig. 4. The secretion of ACTH (panel A) and corticosterone (CORT; panel B) in rats previously

submitted to chronic treatment with vehicle (VEH; 1 ml/kg, s.c. per day) or corticosterone

(CORT; 20 mg/kg, s.c. per day) for 14 days. On day 15, animals were decapitated without any

stress exposure (0 min) or after an acute restraint stress session for 10 and 30 min. Bars represent

the mean and vertical lines denote the standard error of the mean of 6 observations. *** P <

0.001 vs 30 min VEH.

Page 25 of 30



Draft

Table 1. Effect of chronic corticosterone (CORT) treatment as compared to that of VEH and home cage control conditions (CTRL) on total body weight, body weight gain, and relative adrenal gland and thymus weights expressed as mg of tissue per 100 g of body weight. One day after the end of treatments the adrenal glands from both sides and thymus were removed, cleaned of adherent tissue and weighed immediately afterwards.

Body weight (g)Relative organ weight

(mg/100 g body weight)Initial Final Gain LAG RAG Thymus

CTRL 174.3±2.1 274.6±3.2 57.7±1.4 9.08±0.20 8.46±0.25g 321±7.5VEH 174.4±1.7 266.4±3.6a 52.7±1.1c 9.77±0.26 9.44±0.25 287±12.6h

CORT 166.8±2.1 241.1±3.4b 44.6±1.3d 7.08±0.15e 6.58±0.20f 208±8.5ia P < 0.01 vs CTRL Final; b P < 0.0001 vs CTRL Final; c P < 0.001 vs CTRL; d P < 0.0001 vs CTRL;e P < 0.0001 vs CTRL and VEH; f P < 0.0001 vs CTRL and VEH; g P < 0.01 vs CTRL LAG; h P < 0.01 vs VEH; i P < 0.0001 vs CTRL

Page 26 of 30



Draft

Figure 1

185x116mm (150 x 150 DPI)

Page 27 of 30



Draft

Figure 2

264x168mm (300 x 300 DPI)

Page 28 of 30



Draft

Figure 3

90x75mm (300 x 300 DPI)

Page 29 of 30



Draft

Figure 4

199x83mm (300 x 300 DPI)

Page 30 of 30



draft...draft canadian journal of physiology and pharmacology april 2019 effect of chronic...

Documents