Journal of Neuroendocrinology, 2014, 26, 844–852 © 2014 British Society for Neuroendocrinology
ORIGINAL ARTICLE
Suckling and Salsolinol Attenuate Responsiveness of the HypothalamicPituitary-Adrenal Axis to Stress: Focus on Catecholamines, Corticotrophin-Releasing Hormone, Adrenocorticotrophic Hormone, Cortisol and Prolactin Secretion in Lactating Sheep M. Hasiec*, D. Tomaszewska-Zaremba† and T. Misztal* *Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland. †Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland.
Journal of Neuroendocrinology
Correspondence to: Professor Tomasz Misztal, Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3 Street, 05-110 Jablonna, Poland (e-mail: [email protected]).
In mammals, the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to stress is reduced during lactation and this mainly results from suckling by the offspring. The suckling stimulus causes a release of the hypothalamic 1-metyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) (a derivative of dopamine), one of the prolactin-releasing factors. To investigate the involvement of salsolinol in the mechanism suppressing stress-induced HPA axis activity, we conducted a series of experiments on lactating sheep, in which they were treated with two kinds of isolation stress (isolation from the flock with lamb present or absent), combined with suckling and/or i.c.v infusion of salsolinol and 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ; an antagonistic analogue of salsolinol). Additionally, a push–pull perfusion of the infundibular nucleus/median eminence (IN/ME) and blood sample collection with 10-min intervals were performed during the experiments. Concentrations of perfusate corticotrophin-releasing hormone (CRH) and catecholamines (noradrenaline, dopamine and salsolinol), as well as concentrations of plasma adenocorticotrophic hormone (ACTH), cortisol and prolactin, were assayed. A significant increase in perfusate noradrenaline, plasma ACTH and cortisol occurred in response to both kinds of isolation stress. Suckling and salsolinol reduced the stress-induced increase in plasma ACTH and cortisol concentrations. Salsolinol also significantly reduced the stress-induced noradrenaline and dopamine release within the IN/ME. Treatment with 1-MeDIQ under the stress conditions significantly diminished the salsolinol concentration and increased CRH and cortisol concentrations. Stress and salsolinol did not increase the plasma prolactin concentration, in contrast to the suckling stimulus. In conclusion, salsolinol released in nursing sheep may have a suppressing effect on stress-induced HPA axis activity and peripheral prolactin does not appear to participate in its action. Key words: suckling, salsolinol, stress, HPA axis, lactation
Lactation is characterised by many adaptive changes in morphology, physiology and mother’s behaviour to ensure proper care and development of the offspring. The post-partum adaptations include suppressed reproduction, increased appetite, milk production and maternal behaviours (i.e. nursing of newborns) (1). There are also relevant changes in basal and stress-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis, which have a pivotal role in the metabolic demands of lactating females and developing offspring because corticosteroids freely enter maternal milk. Studies
doi: 10.1111/jne.12222
on rats have demonstrated that the circadian rhythm of the corticosterone secretion is flattened during lactation (2). Furthermore, the attenuated stress-induced activity of the HPA axis has been observed in lactating rats (2), human (3) and sheep (4). The modifications of HPA axis activity during lactation are known to largely result from alterations within the central nervous system, first, in the paraventricular nucleus (PVN) of the hypothalamus, where corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are synthesised, and, second, in the noradrenergic
Suckling and salsolinol attenuates HPA axis activity
system, which plays a key role in the regulating its activity (5). During lactation, noradrenaline has lower inputs to neurones in the PVN (6). Moreover, these neurones have a reduced ability to respond to this neurotransmitter (7,8). Besides regulation of CRH and AVP secretion within the PVN, there are also changes in the anterior pituitary, where adenocorticotrophic hormone (ACTH) is synthesised and released. In rats, ACTH secretion following i.v. injection of CRH was lower in lactating rats compared to virgin animals but higher after similar injections of AVP (9). Suckling appears to be a crucial stimulus that triggers the suppression of the HPA axis stress response during lactation. Female rats that were suckled by their pups were reported to have a lower ACTH and cortisol response to stress compared to dams whose nipples were removed and only stayed with their pups (10). Similarly, breastfeeding women had blunted total plasma cortisol and salivary free cortisol responses to psychosocial stress compared to women who only held their infants (11). In sheep, the presence of the lambs appears to be sufficient for the partial attenuation of the HPA axis response to isolation and restrain stress, although suckling enhances the attenuation of cortisol response to this stress (4). Recently, it was reported that suckling causes an increase in 1metyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) (a derivative of dopamine) concentration in the extracellular matrix of the infundibular nucleus/median eminence (IN/ME) in lactating sheep (12). This compound had been generally associated with dysfunction of dopaminergic neurones (13), although salsolinol is now also considered to be a physiological stimulator of prolactin release in rodents (14) and ruminants (12,15). In sheep, elevated prolactin secretion during lactation and suckling-induced prolactin surge appears to result from the stimulatory action of salsolinol on either prolactin mRNA expression (16) or the release of this hormone (12). An abundant amount of salsolinol in the hypothalamus of lactating sheep, its release in response to suckling (12) and the involvement of the hypothalamic neuroendocrine dopaminergic system in the regulatory mechanisms for the release of some pituitary hormones (17,18) led us to investigate whether salsolinol participates in the inhibition of the HPA axis response to stress. To confirm this hypothesis, a series of experiments were performed on lactating sheep, in which the isolation stress of varying strength was used with a combination of suckling and i.c.v. treatments with either salsolinol or its antagonistic analogue 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ).
Materials and methods Animal management The experiment was performed on the Polish Longwool breed of sheep (3– 4 years old; n = 15). The ewes were mated naturally in September and lambed the following February. They were maintained indoors in individual pens under natural lightening conditions (52°N, 21°E). Sheep were fed twice a day with a diet formulated to fulfil all of the National Research Institute of Animal Production recommendations for pregnancy and lactation (Norms 1993), with hay and water available ad lib. During the experiment, nursing sheep were kept in comfortable cages, with or without lamb, where they could lie down. All animal procedures were conducted in accordance with Journal of Neuroendocrinology, 2014, 26, 844–852
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the Polish Guide for the Care and Use of Animals (1997) and were approved by the Local Ethics Committee.
Brain surgery All ewes were bilaterally implanted with two stainless steel guide cannulae: one into the IN/ME; (outer diameter 1.6 mm, positions: frontal 31.0– 32.5 mm and sagittal 1.0 mm) and the second, into the third ventricle of the brain (IIIv, outer diameter 1.2 mm, positions: frontal 29.5–31.0 mm and sagittal 0.3–0.5 mm), in accordance with the stereotaxic coordinate system for the sheep hypothalamus (19). The implantations were performed during the second month of pregnancy under general anaesthesia (xylazine 40 lg/ kg of body mass, i.v., Xylapan; Vetoquinol Biowet, Pulawy, Poland; ketamine 10–20 mg/kg of body mass, i.v., Bioketan; Vetoquinol Biowet), through a drill hole in the skull, in accordance with the procedure described by Traczyk and Przekop (20). The guide cannulae were fixed to the skull with stainless steel screws and dental cement. The external opening to the canal was closed with a stainless steel cap. After surgery, the ewes were injected daily with antibiotics (1 g of streptomycin and 1 200 000 IU benzylpenicillin; Polfa, Warsaw, Poland) for 5 days and with analgesics (metamizole sodium 50 mg/ animal, Biovetalgin; Biowet Drwalew, Drwalew, Poland, or meloxicam 1.5 mg/animal, Metacam; Boehringer Ingelheim, Ingelheim, Germany) for 4 days. The placement of the cannulae was confirmed after slaughtering (after weaning) by the injection of a blue ink.
Experimental design and drug treatments The experiment was performed during the fifth week of lactation, as described in a previous study (12). The isolation stress of varying strengths was used: isolation from the flock with lamb present (Low) and isolation from the flock with lamb absent (High), combined with suckling and the i.c.v. infusion of salsolinol or 1-MeDIQ. Each sheep was used twice at a 3– 4-day interval, forming six experimental groups; No Stress (control); Low Stress (n = 5); Low Stress + Suckling; High Stress (n = 5); High Stress + Salsolinol; Low Stress + 1-MeDIQ (n = 5). The experiment schedule is presented in Table 1. Lambs had limited access to mother’s udder for 1 h before the experiment. In the groups of No Stress, Low Stress, Low Stress + Suckling, Low Stress + 1-MeDiQ, sheep mothers were accompanied by their lambs. Except for the Low Stress + Suckling Group, lambs had no access to the udder during the whole experiment. Both salsolinol and 1-MeDIQ were synthesised and kindly provided by Professor Ferenc Fulop (Institute of Pharmaceutical Chemistry, University of Szeged, Hungary). 1-MeDIQ was chosen as a compound antagonising salsolinol effect because it is a potent inhibitor of salsolinol-, suckling-, and
Table 1. Schedule of the Treatments in the Experimental Groups of Sheep.
Group No stress Low stress Low stress + suckling High stress High stress + salsolinol Low stress + 1-MeDIQ
Accompanying sheep
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1-MeDIQ, 1-methyl-3,4-dihydro-isoqinoline; RLs, Ringer-Locke solution. © 2014 British Society for Neuroendocrinology
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stress- induced prolactin release (21,22) and i.c.v. infusion of this compound reduced salsolinol content in perfusates from the IN/ME, almost to an undetectable level (23). The drugs were dissolved in Ringer-Locke solution (RLs), divided into aliquots and stored at 20 °C. Sheep were treated with RLs alone, salsolinol or 1-MeDIQ solutions in a series of four 30-min infusions into the IIIv, at 30-min intervals. Doses selected on the base of previous studies (12,23,24) were 15 lg/60 ll/30 min (total 60 lg) for salsolinol and 60 lg/60 ll/30 min (total 240 lg) for 1-MeDIQ. A fresh quantity of the drug solution was used each time to ensure stability during the experiment. All infusions were conducted from 10.00 to 14.00 h using a BAS BeeTM microinjection pump (Bioanalitycal System Inc. West Lafayette, IN, USA) and calibrated 1.0-ml gas-tight syringes. Simultaneously, the IN/ME was perfused with the RLs by the push–pull method (12) with a flow rate of 10 ll/min. One hundred microlitres of the perfusion fluid was collected (10 min) to measure salsolinol, noradrenaline and dopamine concentrations and, alternately, 200 ll (in the next 20 min) to measure the CRH concentration. The tubes for perfusates contained 25 ll of 0.1 mM ascorbic acid or 25 ll of aprotinin (5–10 TIU/ml; Sigma-Aldrich, St Louis, MO, USA) to ensure the stability of catecholamines and CRH, respectively. The total perfusion time was 5 h, including a pre-perfusion period from 9.00 to 10.00 h. Immediately the tubes were filled, they were frozen at 80 °C and kept until assayed. All perfusions were conducted using a calibrated 1.0 ml gas-tight syringe and a CMA 402 pump (CMA, Stockholm, Sweden). During the experiment, blood samples were collected every 10 min through a catheter inserted into the jugular vein the day before the experiment. Six millilitres of blood were taken for each sample (the total volume was approximately 150 ml) and immediately divided evenly into two tubes: one with heparin (30 ll, 500 units/ml; Polfa) to measure prolactin and cortisol concentrations and the other with ethylenediaminetetraacetic acid (EDTA) (100 ll, 0.5 M; Sigma-Aldrich) to measure the ACTH concentration. After centrifugation, plasma was stored at 20 °C until the hormones were assayed.
Analytical techniques Catecholamines The concentrations of salsolinol, noradrenaline and dopamine were analysed using high-performance liquid chromatography with electrochemical detection. Perfusates were centrifuged for 15 min and filtered through a 0.22-lm membrane GVWP; 50-ll aliquots of each filtrate were injected into a LC-18DB (inner diameter 15 cm 9 4.6 mm, 5 lm). The Supleco column was protected by a superguard LC-18-DB 5 lm Supercosil, 2-cm precolumn (Sigma-Aldrich). The column was coupled with an electrochemical detector (HP 1049 A Programmable Electrochemical Detector; Hewlett Packard, Santa Clara, CA, USA) equipped with a glassy carbon working electrode and Ag/ AgCl reference electrode. The oxidative potential of the electrochemical detector was 0.650 V. Samples were eluted isocratically with a mobile phase consisting of 0.01 mol/l NaCl, 0.001 mol/l EDTA, 53–55 mg/l octal sulphate sodium salt and 12% CH3OH; the pH of the mobile phase was 3.6. The mobile phase was filtered through a 0.22-lm GVWP membrane and degassed under vacuum with ultrasonic agitation. The flow rate was 0.8 ml/ min. Stock solutions of the standard were stored at 20 °C. The limits of detection were 10 pg/50 ll for salsolinol and 5 pg/50 ll for noradrenaline and dopamine.
Hormones The concentrations of cortisol and prolactin in plasma were assayed by radioimmunoassay (RIA). The cortisol was measured according to Kokot and © 2014 British Society for Neuroendocrinology
Stupnicki (25) using rabbit anti-cortisol antisera (R/75) and cortisol standard (Sigma-Aldrich). The assay sensitivity was 0.95 ng/ml and the intra- and inter-assay coefficients of variation for cortisol were 10% and 12%, respectively. The concentration of prolactin in the plasma was assayed using antiovine prolactin and antirabbit c-globulin antisera as described by Wolinska et al. (26). The prolactin standard was synthesised and kindly provided by Professor Kazimierz Kochman from our institute. The assay sensitivity for prolactin was 2 ng/ml and the intra- and inter-assay coefficients of variation were 9% and 12%, respectively. The concentrations of plasma ACTH and perfusate CRH were determined using commercial RIA kits (DiaSorin, Stillwater, MN, USA, catalogue number 24130 for ACTH; Bachem, San Carlos, CA, USA, catalogue number S-2108 for CRH). Ranges of the calibrated curve were 20–500 pg/ml for the ACTH assay and 10–1280 pg/ml for the CRH assay. Perfusates for the CRH determination were freeze-dried and then dissolved in 120 ll of RIA buffer to concentrate the samples within the measuring range.
Statistical analysis The significance of differences in ACTH, cortisol and prolactin concentrations among all the experimental groups was examined by one-way ANOVA followed by the least significance differences post-hoc test (STATISTICA; StatSoft, Inc., Tulsa, OK, USA). For nonparametric statistics, the Kruskal–Wallis test followed by multiple comparisons of average ranks for all tests was used to determine the significance of the differences in CRH, salsolinol, noradrenaline and dopamine concentrations among the experimental groups. All data are expressed as the mean SEM.
Results Exposition to stress Both low and high stress evoked a significant (P < 0.001) increase in the concentration of plasma components of the HPA axis (i.e. ACTH and cortisol), whereas no considerable stress-induced changes were observed in the perfusate CRH level. A significant (P < 0.001) increase in noradrenaline concentration was noted in all stressed animals, with the highest value in the High Stress group. Also dopamine concentration increased clearly in both stressed groups, compared to controls, in which dopamine was at an undetectable level. No significant differences were found in salsolinol concentration amongst the Non Stress, Low Stress and High Stress groups. The prolactin concentration even decreased significantly (P < 0.01) in the Low Stress group in comparison with the No Stress group. A clear response to both kinds of stress was also observed in the profiles of ACTH and cortisol secretion, whereas the prolactin profile is similar in control and stressed animals (Figs 1A, 2A and 3A, respectively).
Exposition to low stress and effect of suckling Suckling significantly (P < 0.001) reduced the ACTH and cortisol responses to stress to a similar level as in the No Stress group. No significant changes in CRH level were observed in the Low Stress + Suckling group, compared to the No Stress and Low Stress groups, although, visually, the perfusate CRH concentration in this first was the lowest. Noradrenaline and dopamine concentrations, Journal of Neuroendocrinology, 2014, 26, 844–852
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Fig. 1. Mean plasma adrenocotricotrophic hormone (ACTH) concentrations in consecutive blood samples in accordance with the type of treatment. (A) Exposition to stress (groups: No stress, Low Stress, High Stress). (B) Exposition to low stress and effect of suckling (groups: No Stress, Low Stress, Low Stress + Suckling). (C) Exposition to high stress and effect of salsolinol (groups: No Stress, High Stress, High Stress + Salsolinol). (D) Exposition to low stress and effect of 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ) (No Stress, Low Stress, Low Stress + 1-MeDIQ).
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Salsolinol infused i.c.v. significantly reduced the ACTH (P < 0.001) and cortisol (P < 0.05) responses to high stress but did not affect the perfusate CRH concentration. However, noradrenaline and dopamine concentrations decreased significantly (P < 0.001) in sheep exposed to high stress, following salsolinol infusion, in comparison with the sheep only stressed. Although, the concentration of salsolinol in the High Stress + Salsolinol group was above the range of analysis, prolactin was even lower than that in the No Stress group. A reduction of the HPA axis response to stress by salsolinol is clearly pronounced in the profile of ACTH secretion (Fig. 1C). Cortisol release returned to the normal rhythm after the first burst following the stress exposition (Fig. 2C).
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although increased in stressed animals, were not changed statistically by suckling. However, suckling significantly (P < 0.001) increased salsolinol and prolactin concentration compared to the Journal of Neuroendocrinology, 2014, 26, 844–852
1-MeDIQ treatment of the Low Stress group significantly (P < 0.001) raised CRH and cortisol concentration, compared to the No Stress and only Low Stress groups. In case of ACTH, the concentration noted in the Low Stress + 1-MeDIQ group was significantly (P < 0.001) lower than in the Low Stress group but still significantly (P < 0.01) higher than in the No Stress group. 1-MeDIQ infused i.c.v. did not change the noredrenaline, dopamine and prolactin concentrations significantly, compared to the Low Stress group, but reduced the salsolinol level below the assay detection limit. The changes in the ACTH and cortisol concentrations, or their lack as in case of prolactin, are clearly reflected by the profiles of secretion, monitored during the experiment (Figs 1D, 2D and 3D, respectively). The mean concentrations of each studied catecholamine and hormone are shown in Table 2, in accordance with the type of treatments and with a single threshold for statistical significance (P < 0.05).
Discussion The present study extends on existing knowledge concerning the attenuation of the stress response of the HPA axis in lactating sheep and, for the first time, demonstrates the central action of salsolinol in this process. We used two experimental in vivo techniques (i.e. i.c.v. infusion, combined with the push–pull perfusion of © 2014 British Society for Neuroendocrinology
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Cortisol (ng/ml)
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Fig. 2. Mean plasma cortisol concentrations in consecutive blood samples collected in accordance with the type of treatment. (A) Exposition to stress (groups: No stress, Low Stress, High Stress); (B) Exposition to low stress and suckling effect (groups: No Stress, Low Stress, Low Stress + Suckling). (C) Exposition to high stress and salsolinol effect (groups: No Stress, High Stress, High Stress + Salsolinol). (D) Exposition to low stress and 1-methyl3,4-dihydro-isoqinoline (1-MeDIQ) effect (No Stress, Low Stress, Low Stress + 1-MeDIQ).
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the IN/ME) for simultaneous administration of the drug and identification of the substances released from the nerve terminals into the hypophyseal portal system. Besides direct cannulation of the © 2014 British Society for Neuroendocrinology
hypophyseal portal vessels (27), the push–pull perfusion of the IN/ ME is a useful technique, providing information about neural factors, neurotransmitters and neurohormones reaching the anterior pituitary (28,29). Changes in both ACTH and cortisol secretion are the most common plasma measures that indicate the HPA axis response to stress. In the present study, plasma ACTH and cortisol concentrations in lactating sheep increased significantly in response to isolation from the flock, either with lambs present but unable to suckle or when lambs were also isolated. Interestingly, there were no differences in the strength of the stress response between these two groups as a result of similar mean ACTH and cortisol concentrations. This is contrary to the findings of the study by Tilbrook et al. (4) in lactating sheep, which showed that the reaction to isolation and restrain stress was higher in sheep isolated from lambs than in sheep with lambs present but unable to suckle. By contrast to plasma components of the HPA axis, stress did not affect the CRH concentration within the IN/ME, whereas noradrenaline release in this site intensified depending on the strength of the stress. Our data suggest that, during lactation, another hypothalamic factor could be more potent in stimulating ACTH release in response to stress. Because the ACTH response to AVP increased in lactating rats compared to virgins (9), this neuropeptide is the most likely candidate. Moreover, the lack of CRH response to a given type of stress might result from a lesser sensitivity of the PVN to the noradrenergic input during lactation, as described in rats (8). The suckling stimulus is assigned a crucial inhibiting role in the stress-induced activity of the HPA axis during lactation (8) and the present study strongly confirmed this phenomenon. As expected, suckling by the lambs significantly suppressed an increase in both plasma ACTH and cortisol concentration in response to isolation stress. This is consistent with the earlier study, where a greater cortisol response to isolation and restrain stress occurred in sheep that stayed with their lamb unable to suckle than in sheep whose lambs had free access to the udder (4). However, again, there is a discrepancy between the present study and the study by Tilbrook et al. (4) in terms of plasma ACTH concentration because, in the latter study, there were no differences in ACTH concentration between these two groups. We observed a significantly lower plasma concentration of ACTH in stressed sheep suckled by their lamb compared to sheep whose lambs had restricted access to the udder. This might be a result of differences in an experimental set-up such as the period of lactation used or the fact that, in the present study, all lambs had a 1-h limited access to the udder before the actual experiment. The attenuation phenomenon of the stress response of the HPA axis by suckling may partly result from the action of salsolinol. Our Journal of Neuroendocrinology, 2014, 26, 844–852
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Fig. 3. Mean plasma prolactin concentrations in consecutive blood samples in accordance with the type of treatment. (A) Exposition to stress (groups: No stress, Low Stress, High Stress). (B) Exposition to low stress and suckling effect (groups: No Stress, Low Stress, Low Stress + suckling). (C) Exposition to high stress and salsolinol effect (groups: No Stress, High Stress, High Stress + Salsolinol). (D) Exposition to low stress and 1-methyl-3,4-dihydroisoqinoline (1-MeDIQ) effect (No Stress, Low Stress, Low Stress + 1-MeDIQ).
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earlier studies showed that this dopamine-derived compound is present in the IN/ME of lactating sheep and its concentration increases during suckling (12). This is also confirmed in the present Journal of Neuroendocrinology, 2014, 26, 844–852
study. Moreover, we have shown that salsolinol infused into the IIIv inhibited the stress-induced release of peripheral components of the HPA axis (i.e. ACTH and cortisol), although it had no effect on the CRH concentration. In turn, the use of a structural analogue of salsolinol (i.e. 1-MeDIQ), known to be an inhibitor of some of the actions of salsolinol (21,23), provided some unexpected data. The infusion of 1-MeDIQ into the IIIv caused an exceptional increase in CRH concentration in perfusates collected from the IN/ME, compared to the other experimental groups. Because 1-MeDIQ completely excluded salsolinol from the extracellular matrix of the IN/ ME, our data suggest that salsolinol, indeed, could play a meaningful role in inhibiting CRH release during lactation. This point of view, however, is not reflected by an increase in the mean plasma ACTH concentration of lightly stressed sheep, in which 1-MeDIQ was expected to potentiate the stress response as a whole. However, the same sheep had the highest plasma cortisol concentration, compared to all the other groups, and so a possible explanation could be the strong feedback of cortisol on ACTH in this group. The action of 1-MeDIQ by itself on corticotrophs and adrenal cortex cells cannot be excluded, although it is less probable because the peripheral injection of 1-MeDIQ did not attenuate corticosterone response to immobilisation stress in male rats (21). The detailed central mechanism for the action of salsolinol on the individual components of the HPA axis is not clear. The present study revealed a possible dual effect of this isoquinoline. First, because a meaningful reduction of salsolinol within the IN/ME by 1-MeDIQ caused a more than three-fold increase of CRH concentration, salsolinol appears to be a major inhibiting factor of the stress-induced CRH release in lactating sheep. Second, salsolinol at a higher concentration, following suckling or infusion, reduced the stress-induced noradrenaline release. This neurotransmitter released within the PVN from the brainstem noradrenergic nerve terminals is known to be crucial for activation of the HPA axis by stress (30). During lactation, changes in this system are partly accountable for the reduced HPA axis response to stress (6). The reduced noradrenergic activity is associated with the suckling stimulus because females separated from their pups 48 h earlier have a higher basal noradrenaline concentration in the PVN than suckled females (5). In the present study, we measured the noradrenaline concentration within the extracellular matrix of the IN/ME, in addition to CRH. Thus, the concentration of noradrenaline derived from this site may not fully reflect the amount of this neurotransmitter reaching the PVN but rather the amount delivering to the anterior pituitary. Noradrenaline may also possibly affect the HPA axis acting directly on the pituitary corticotrophs. b-adrenergic receptors were found in the anterior pituitary of several species (31,32) and noradrenergic nerve terminals were located in the ovine infundibular nucleus (33). © 2014 British Society for Neuroendocrinology
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Table 2. The Mean Concentrations of Catecholamines (Noradrenaline, Dopamine, Salsolinol) and Hormones [Corticotrophin-Releasing Hormone (CRH), Adenocorticotrophic Hormone (ACTH), Cortisol, Prolactin] in Accordance with the Type of Treatment: Exposition to Stress (Groups: No Stress, Low Stress, High Stress); Exposition to Low Stress and Effect of Suckling (Groups: No Stress, Low Stress, Low Stress + Suckling); Exposition to High Stress and Effect of Salsolinol (Groups: No Stress, High Stress, High Stress + Salsolinol); and Exposition to Low Stress and Effect of 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ) (Groups: No Stress, Low Stress, Low Stress + 1-MeDIQ). HPA axis
Group
CRH (pg/ml)
Catecholamines ACTH (pg/ml)
Exposition to stress No stress 35.61 4.30 59.73 Low stress 40.82 3.38 97.19 High stress 42.22 6.79 101.13 Exposition to low stress and effect of suckling No stress 35.61 4.30 59.73 Low stress 40.82 3.38 97.19 Low stress + suckling 32.04 3.29 50.90 Exposition to high stress and effect of salsolinol No stress 35.61 4.30 59.73 High stress 42.22 6.79 101.13 High stress + salsolinol 48.49 5.54 58.79 Exposition to low stress and effect of 1-MeDIQ 59.73 No stress 35.61 4.30a Low stress 40.82 3.38a 97.19 Low stress + 1-MeDIQ 134.74 15.26b 74.94
Cortisol (ng/ml)
Noradrenaline (pg/50 ll)
Dopamine (pg/50 ll)
Salsolinol (pg/50 ll)
Prolactin (ng/ml)
2.48a 5.09b 6.80b
18.52 1.23a 29.20 1.56b 26.21 1.61b
41.71 2.51a 89.97 6.72b 139.49 7.00c
Below detection 96.59 16.17 114.65 14.25
23.78 4.48 32.39 6.10 32.44 3.88
110.79 8.45a 77.79 7.81b 89.23 5.08
2.48a 5.09b 1.75a
18.52 1.23a 29.20 1.56b 14.03 0.87a
41.71 2.51a 89.97 6.72b 66.78 5.68b
Below detection 96.59 16.17 50.81 5.27
23.78 4.48a 32.39 6.10a 227.49 11.47b
110.79 8.45a 77.79 7.81b 276.16 14.18c
2.48a 6.80b 1.82a
18.52 1.23a 26.21 1.61b 20.55 1.09a
41.71 2.51a 139.49 7.00b 57.22 4.78a
Below detection 114.65 14.25b 44.11 10.19a
23.78 4.48 32.44 3.88 Above detection
110.79 8.45a 89.23 5.08 73.02 2.81b
2.48a 5.09c 2.74b
18.52 1.23a 29.20 1.56b 38.20 2.60c
41.71 2.51a 89.97 6.72b 80.91 7.04b
Below detection 96.59 16.17 65.28 6.53
23.78 4.48 32.39 6.10 Below detection
110.79 8.45a 77.79 7.81b 93.18 4.93
HPA, hypothalamic-pituitary-adrenal. Different superscript lowercase letters indicate a statistically significant difference (P < 0.05).
Moreover, noradrenaline stimulated the release of ACTH from cultured ovine anterior pituitary cells (34). Regardless of the pathway for the action of salsolinol, the inhibitory mechanism for stressinduced activity of the HPA axis in nursing females could also be a consequence of reduced noradrenaline release. This is in line with the latest research showing that salsolinol inhibits tyrosine hydroxylase (the rate-limiting enzyme of the catecholamines biosynthesis) activity (35). Because salsolinol is known as a prolactin-releasing factor (36) and prolactin receptors are present in the hypothalamus and other regions of the central nervous system (37), this hormone appears to mediate the discussed phenomenon of the action of salsolinol on the HPA axis. In the present study, stress was an inefficient stimulus for increasing the plasma prolactin concentration in contrast to suckling. It might be a result of stress-induced dopamine release within the IN/ME, which could prevent an additional increase in stress-induced prolactin. Interestingly, although the infusion of salsolinol caused an inhibition of ACTH-cortisol response to high stress, this compound was unable to trigger the release of extra prolactin into circulation. These data suggest that salsolinol is ineffective as a prolactin-releasing factor in lactating sheep during stressful conditions and it is unlikely that prolactin mediates the inhibitory effect of salsolinol on the stress-induced activity of the HPA axis. Therefore, another factor(s) should be taken into account in suckling stimulation of prolactin release under these unfavourable conditions and oxytocin may be one of the candidates. © 2014 British Society for Neuroendocrinology
Evidence exists suggesting that oxytocin mediates the prolactin surge induced by suckling (38) and, moreover, is able to suppress the cortisol response to an audiovisual stressor in nonlactating and lactating sheep (39). This, however, is beyond the scope of the present study and requires further investigation. In summary, our research points to the profound changes with respect to regulating the stress response that occurs during lactation and the relevant role of salsolinol at this physiological stage. The reported results suggest that salsolinol mediates the inhibiting effect of suckling on stress-induced HPA axis activity in lactating sheep. A better understanding of the molecular action of salsolinol is necessary to confirm this and explain precisely the physiological actions of this compound.
Acknowledgements This research was supported by the National Science Centre, Poland, Grant number N N311 082037. The authors would like to thank Professor F. Fulop (Szeged University, Hungary) for preparing and providing salsolinol and 1-MeDIQ; veterinary surgeon J. Rutkowski for help with the brain surgery; K. Romanowicz PhD for help with the hormone RIA; K. Gorski PhD and E. Marciniak MSc for help with blood sampling; and Mr W. Mrozek for animal care. The authors have no conflicts of interest to declare.
Received 22 November 2013, revised 12 April 2014, accepted 30 August 2014 Journal of Neuroendocrinology, 2014, 26, 844–852
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18 Olah M, Feher P, Ihm Z, Bacskay I, Kiss T, Freeman ME, Nagy GM, Vecsernyes M. Dopamine-regulated adrenocorticotropic hormone secretion in lactating rats: functional plasticity of melanotropes. Neuroendocrinology 2009; 90: 391–401. 19 Welento J, Szteyn S, Milart Z. Observations on the stereotaxic configuration of the hypothalamus nuclei in the sheep. Anat Anz 1969; 124: 1–27. 20 Traczyk W, Przekop F. Methods of investigation of the function of the hypothalamus and hypophysis in chronic experiments in sheep. Acta Physiol Pol 1963; 14: 217–226. 21 Bodnar I, Mravec B, Kubovcakova L, Toth EB, Fulop F, Fekete MIK, Kvetnansky R, Nagy GM. Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotropic prolactin-releasing factor, salsolinol. J Neuroendocrinol 2004; 16: 208–213. 22 Radnai B, Mravec B, Bodnar I, Kubovcakova L, Fulop F, Fekete MI. Pivotal role of an endogenous tetrahydroisoquinoline, salsolinol, in stress- and suckling-induced release of prolactin. Ann NY Acad Sci 2004; 1018: 183–191. 23 Misztal T, Gorski K, Tomaszewska-Zaremba D, Fulop F, Romanowicz K. Effects of a structural analogue of salsolinol, 1-MeDIQ, on pituitary prolactin release and dopaminergic activity in the mediobasal hypothalamus in nursing sheep. Brain Res 2010; 1307: 72–77. 24 Gorski K, Romanowicz K, Herman A, Molik E, Gajewska A, TomaszewskaZaremba D, Misztal T. The possible involvement of salsolinol and hypothalamic prolactin in the central regulatory processes in ewes during lactation. Reprod Domest Anim 2010; 45: e54–e60. 25 Kokot F, Stupnicki R. Metody radioimmunologiczne i radiokompetycyjne stosowane w klinice [in polish]. Warsaw: PZWL, 1985. 26 Wolinska E, Polkowska J, Domanski E. The hypothalamic centres involved in the control of production and release of prolactin in sheep. J Endocrinol 1977; 73: 21–29. 27 Caraty A, Locatelli A, Moenter SM, Karsch FJ. Sampling of hypophyseal portal blood of conscious sheep for direct monitoring of hypothalamic neurosecretory substances. In: Levine JE, ed. Pulsatility in Neuroendocrine Systems. Methods in Neurosciences, Vol. 20. New York, NY: Academic Press, 1994: 162–183. 28 Domanski E, Chomicka LK, Ostrowska A, Gajewska A, Mateusiak K. Releasing of luteinizing hormone-releasing hormone, b-endorphin and noradrenaline by the nucleus infundibularis/median eminence during periovulatory period in the sheep. Neuroendocrinology 1991; 54: 151– 158. 29 Tomaszewska D, Przekop F. Catecholaminergic activity in the medial preoptic area and nucleus infundibularis-median eminence of anestrous ewes in normal physiological state and under stress condition. J Neural Transm 1999; 106: 1031–1043. 30 Pacak K, Palkovits M, Kopin IJ, Goldstein DS. Stress-induced norepinephrine release in the hypothalamic paraventricular nucleus and pituitary; adrenocortical and sympathoadrenal activity: in vivo microdialysis studies. Front Neuroendocrinol 1995; 19: 89–150. 31 De Souza EB. Beta-2-adrenergic receptors in pituitary. Identification, characterization, and autoradiographic localization. Neuroendocrinology 1985; 41: 289–296. 32 Sato M, Kubota Y, Malbon CC, Tohyama M. Immunohistochemical evidence that most rat corticotrophs contain beta-adrenergic receptors. Neuroendocrinology 1989; 50: 577–583. 33 Tillet Y, Thibault J. Catecholamine-containing neurons in the sheep brainstem and diencephalon: immunohistochemical study with tyrosine hydroxylase (TH) and dopamine-b-hydroxylase antibodies. J Comp Neurol 1989; 290: 69–104. 34 Liu JP, Clarke IJ, Funder JW, Engler D. Evidence that the central noradrenergic and adrenergic pathways activate the hypothalamic-pituitaryadrenal axis in the sheep. Endocrinology 1991; 129: 200–209.
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35 Briggs GB, Nagy GM, Dickson PW. Mechanism of action of salsolinol on tyrosine hydroxylase. Neurochem Int 2013; 63: 726–731. 36 Mravec B. Salsolinol, a derivate of dopamine, is a possible modulator of catecholaminergic transmission: a review of recent developments. Physiol Res 2006; 55: 353–364. 37 Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev 2000; 80: 1523–1631.
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38 Kennett JE, Poletini MO, Fitch CA, Freeman ME. Antagonism of oxytocin prevents suckling- and estradiol-induced, but not progesterone-induced, secretion of prolactin. Endocrinology 2009; 150: 2292–2299. 39 Cook CJ. Oxytocin and prolactin suppress cortisol responses to acute stress in both lactating and non-lactating sheep. J Dairy Res 1997; 64: 327–339.
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ORIGINAL ARTICLE
Suckling and Salsolinol Attenuate Responsiveness of the HypothalamicPituitary-Adrenal Axis to Stress: Focus on Catecholamines, Corticotrophin-Releasing Hormone, Adrenocorticotrophic Hormone, Cortisol and Prolactin Secretion in Lactating Sheep M. Hasiec*, D. Tomaszewska-Zaremba† and T. Misztal* *Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland. †Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland.
Journal of Neuroendocrinology
Correspondence to: Professor Tomasz Misztal, Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3 Street, 05-110 Jablonna, Poland (e-mail: [email protected]).
In mammals, the responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to stress is reduced during lactation and this mainly results from suckling by the offspring. The suckling stimulus causes a release of the hypothalamic 1-metyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) (a derivative of dopamine), one of the prolactin-releasing factors. To investigate the involvement of salsolinol in the mechanism suppressing stress-induced HPA axis activity, we conducted a series of experiments on lactating sheep, in which they were treated with two kinds of isolation stress (isolation from the flock with lamb present or absent), combined with suckling and/or i.c.v infusion of salsolinol and 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ; an antagonistic analogue of salsolinol). Additionally, a push–pull perfusion of the infundibular nucleus/median eminence (IN/ME) and blood sample collection with 10-min intervals were performed during the experiments. Concentrations of perfusate corticotrophin-releasing hormone (CRH) and catecholamines (noradrenaline, dopamine and salsolinol), as well as concentrations of plasma adenocorticotrophic hormone (ACTH), cortisol and prolactin, were assayed. A significant increase in perfusate noradrenaline, plasma ACTH and cortisol occurred in response to both kinds of isolation stress. Suckling and salsolinol reduced the stress-induced increase in plasma ACTH and cortisol concentrations. Salsolinol also significantly reduced the stress-induced noradrenaline and dopamine release within the IN/ME. Treatment with 1-MeDIQ under the stress conditions significantly diminished the salsolinol concentration and increased CRH and cortisol concentrations. Stress and salsolinol did not increase the plasma prolactin concentration, in contrast to the suckling stimulus. In conclusion, salsolinol released in nursing sheep may have a suppressing effect on stress-induced HPA axis activity and peripheral prolactin does not appear to participate in its action. Key words: suckling, salsolinol, stress, HPA axis, lactation
Lactation is characterised by many adaptive changes in morphology, physiology and mother’s behaviour to ensure proper care and development of the offspring. The post-partum adaptations include suppressed reproduction, increased appetite, milk production and maternal behaviours (i.e. nursing of newborns) (1). There are also relevant changes in basal and stress-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis, which have a pivotal role in the metabolic demands of lactating females and developing offspring because corticosteroids freely enter maternal milk. Studies
doi: 10.1111/jne.12222
on rats have demonstrated that the circadian rhythm of the corticosterone secretion is flattened during lactation (2). Furthermore, the attenuated stress-induced activity of the HPA axis has been observed in lactating rats (2), human (3) and sheep (4). The modifications of HPA axis activity during lactation are known to largely result from alterations within the central nervous system, first, in the paraventricular nucleus (PVN) of the hypothalamus, where corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are synthesised, and, second, in the noradrenergic
Suckling and salsolinol attenuates HPA axis activity
system, which plays a key role in the regulating its activity (5). During lactation, noradrenaline has lower inputs to neurones in the PVN (6). Moreover, these neurones have a reduced ability to respond to this neurotransmitter (7,8). Besides regulation of CRH and AVP secretion within the PVN, there are also changes in the anterior pituitary, where adenocorticotrophic hormone (ACTH) is synthesised and released. In rats, ACTH secretion following i.v. injection of CRH was lower in lactating rats compared to virgin animals but higher after similar injections of AVP (9). Suckling appears to be a crucial stimulus that triggers the suppression of the HPA axis stress response during lactation. Female rats that were suckled by their pups were reported to have a lower ACTH and cortisol response to stress compared to dams whose nipples were removed and only stayed with their pups (10). Similarly, breastfeeding women had blunted total plasma cortisol and salivary free cortisol responses to psychosocial stress compared to women who only held their infants (11). In sheep, the presence of the lambs appears to be sufficient for the partial attenuation of the HPA axis response to isolation and restrain stress, although suckling enhances the attenuation of cortisol response to this stress (4). Recently, it was reported that suckling causes an increase in 1metyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) (a derivative of dopamine) concentration in the extracellular matrix of the infundibular nucleus/median eminence (IN/ME) in lactating sheep (12). This compound had been generally associated with dysfunction of dopaminergic neurones (13), although salsolinol is now also considered to be a physiological stimulator of prolactin release in rodents (14) and ruminants (12,15). In sheep, elevated prolactin secretion during lactation and suckling-induced prolactin surge appears to result from the stimulatory action of salsolinol on either prolactin mRNA expression (16) or the release of this hormone (12). An abundant amount of salsolinol in the hypothalamus of lactating sheep, its release in response to suckling (12) and the involvement of the hypothalamic neuroendocrine dopaminergic system in the regulatory mechanisms for the release of some pituitary hormones (17,18) led us to investigate whether salsolinol participates in the inhibition of the HPA axis response to stress. To confirm this hypothesis, a series of experiments were performed on lactating sheep, in which the isolation stress of varying strength was used with a combination of suckling and i.c.v. treatments with either salsolinol or its antagonistic analogue 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ).
Materials and methods Animal management The experiment was performed on the Polish Longwool breed of sheep (3– 4 years old; n = 15). The ewes were mated naturally in September and lambed the following February. They were maintained indoors in individual pens under natural lightening conditions (52°N, 21°E). Sheep were fed twice a day with a diet formulated to fulfil all of the National Research Institute of Animal Production recommendations for pregnancy and lactation (Norms 1993), with hay and water available ad lib. During the experiment, nursing sheep were kept in comfortable cages, with or without lamb, where they could lie down. All animal procedures were conducted in accordance with Journal of Neuroendocrinology, 2014, 26, 844–852
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the Polish Guide for the Care and Use of Animals (1997) and were approved by the Local Ethics Committee.
Brain surgery All ewes were bilaterally implanted with two stainless steel guide cannulae: one into the IN/ME; (outer diameter 1.6 mm, positions: frontal 31.0– 32.5 mm and sagittal 1.0 mm) and the second, into the third ventricle of the brain (IIIv, outer diameter 1.2 mm, positions: frontal 29.5–31.0 mm and sagittal 0.3–0.5 mm), in accordance with the stereotaxic coordinate system for the sheep hypothalamus (19). The implantations were performed during the second month of pregnancy under general anaesthesia (xylazine 40 lg/ kg of body mass, i.v., Xylapan; Vetoquinol Biowet, Pulawy, Poland; ketamine 10–20 mg/kg of body mass, i.v., Bioketan; Vetoquinol Biowet), through a drill hole in the skull, in accordance with the procedure described by Traczyk and Przekop (20). The guide cannulae were fixed to the skull with stainless steel screws and dental cement. The external opening to the canal was closed with a stainless steel cap. After surgery, the ewes were injected daily with antibiotics (1 g of streptomycin and 1 200 000 IU benzylpenicillin; Polfa, Warsaw, Poland) for 5 days and with analgesics (metamizole sodium 50 mg/ animal, Biovetalgin; Biowet Drwalew, Drwalew, Poland, or meloxicam 1.5 mg/animal, Metacam; Boehringer Ingelheim, Ingelheim, Germany) for 4 days. The placement of the cannulae was confirmed after slaughtering (after weaning) by the injection of a blue ink.
Experimental design and drug treatments The experiment was performed during the fifth week of lactation, as described in a previous study (12). The isolation stress of varying strengths was used: isolation from the flock with lamb present (Low) and isolation from the flock with lamb absent (High), combined with suckling and the i.c.v. infusion of salsolinol or 1-MeDIQ. Each sheep was used twice at a 3– 4-day interval, forming six experimental groups; No Stress (control); Low Stress (n = 5); Low Stress + Suckling; High Stress (n = 5); High Stress + Salsolinol; Low Stress + 1-MeDIQ (n = 5). The experiment schedule is presented in Table 1. Lambs had limited access to mother’s udder for 1 h before the experiment. In the groups of No Stress, Low Stress, Low Stress + Suckling, Low Stress + 1-MeDiQ, sheep mothers were accompanied by their lambs. Except for the Low Stress + Suckling Group, lambs had no access to the udder during the whole experiment. Both salsolinol and 1-MeDIQ were synthesised and kindly provided by Professor Ferenc Fulop (Institute of Pharmaceutical Chemistry, University of Szeged, Hungary). 1-MeDIQ was chosen as a compound antagonising salsolinol effect because it is a potent inhibitor of salsolinol-, suckling-, and
Table 1. Schedule of the Treatments in the Experimental Groups of Sheep.
Group No stress Low stress Low stress + suckling High stress High stress + salsolinol Low stress + 1-MeDIQ
Accompanying sheep
Accompanying lambs
+
+ + +
+
Suckling
Infusion
N
+
RLs RLs RLs
5 5 5
RLs Salsolinol
5 5
1-MeDIQ
5
1-MeDIQ, 1-methyl-3,4-dihydro-isoqinoline; RLs, Ringer-Locke solution. © 2014 British Society for Neuroendocrinology
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stress- induced prolactin release (21,22) and i.c.v. infusion of this compound reduced salsolinol content in perfusates from the IN/ME, almost to an undetectable level (23). The drugs were dissolved in Ringer-Locke solution (RLs), divided into aliquots and stored at 20 °C. Sheep were treated with RLs alone, salsolinol or 1-MeDIQ solutions in a series of four 30-min infusions into the IIIv, at 30-min intervals. Doses selected on the base of previous studies (12,23,24) were 15 lg/60 ll/30 min (total 60 lg) for salsolinol and 60 lg/60 ll/30 min (total 240 lg) for 1-MeDIQ. A fresh quantity of the drug solution was used each time to ensure stability during the experiment. All infusions were conducted from 10.00 to 14.00 h using a BAS BeeTM microinjection pump (Bioanalitycal System Inc. West Lafayette, IN, USA) and calibrated 1.0-ml gas-tight syringes. Simultaneously, the IN/ME was perfused with the RLs by the push–pull method (12) with a flow rate of 10 ll/min. One hundred microlitres of the perfusion fluid was collected (10 min) to measure salsolinol, noradrenaline and dopamine concentrations and, alternately, 200 ll (in the next 20 min) to measure the CRH concentration. The tubes for perfusates contained 25 ll of 0.1 mM ascorbic acid or 25 ll of aprotinin (5–10 TIU/ml; Sigma-Aldrich, St Louis, MO, USA) to ensure the stability of catecholamines and CRH, respectively. The total perfusion time was 5 h, including a pre-perfusion period from 9.00 to 10.00 h. Immediately the tubes were filled, they were frozen at 80 °C and kept until assayed. All perfusions were conducted using a calibrated 1.0 ml gas-tight syringe and a CMA 402 pump (CMA, Stockholm, Sweden). During the experiment, blood samples were collected every 10 min through a catheter inserted into the jugular vein the day before the experiment. Six millilitres of blood were taken for each sample (the total volume was approximately 150 ml) and immediately divided evenly into two tubes: one with heparin (30 ll, 500 units/ml; Polfa) to measure prolactin and cortisol concentrations and the other with ethylenediaminetetraacetic acid (EDTA) (100 ll, 0.5 M; Sigma-Aldrich) to measure the ACTH concentration. After centrifugation, plasma was stored at 20 °C until the hormones were assayed.
Analytical techniques Catecholamines The concentrations of salsolinol, noradrenaline and dopamine were analysed using high-performance liquid chromatography with electrochemical detection. Perfusates were centrifuged for 15 min and filtered through a 0.22-lm membrane GVWP; 50-ll aliquots of each filtrate were injected into a LC-18DB (inner diameter 15 cm 9 4.6 mm, 5 lm). The Supleco column was protected by a superguard LC-18-DB 5 lm Supercosil, 2-cm precolumn (Sigma-Aldrich). The column was coupled with an electrochemical detector (HP 1049 A Programmable Electrochemical Detector; Hewlett Packard, Santa Clara, CA, USA) equipped with a glassy carbon working electrode and Ag/ AgCl reference electrode. The oxidative potential of the electrochemical detector was 0.650 V. Samples were eluted isocratically with a mobile phase consisting of 0.01 mol/l NaCl, 0.001 mol/l EDTA, 53–55 mg/l octal sulphate sodium salt and 12% CH3OH; the pH of the mobile phase was 3.6. The mobile phase was filtered through a 0.22-lm GVWP membrane and degassed under vacuum with ultrasonic agitation. The flow rate was 0.8 ml/ min. Stock solutions of the standard were stored at 20 °C. The limits of detection were 10 pg/50 ll for salsolinol and 5 pg/50 ll for noradrenaline and dopamine.
Hormones The concentrations of cortisol and prolactin in plasma were assayed by radioimmunoassay (RIA). The cortisol was measured according to Kokot and © 2014 British Society for Neuroendocrinology
Stupnicki (25) using rabbit anti-cortisol antisera (R/75) and cortisol standard (Sigma-Aldrich). The assay sensitivity was 0.95 ng/ml and the intra- and inter-assay coefficients of variation for cortisol were 10% and 12%, respectively. The concentration of prolactin in the plasma was assayed using antiovine prolactin and antirabbit c-globulin antisera as described by Wolinska et al. (26). The prolactin standard was synthesised and kindly provided by Professor Kazimierz Kochman from our institute. The assay sensitivity for prolactin was 2 ng/ml and the intra- and inter-assay coefficients of variation were 9% and 12%, respectively. The concentrations of plasma ACTH and perfusate CRH were determined using commercial RIA kits (DiaSorin, Stillwater, MN, USA, catalogue number 24130 for ACTH; Bachem, San Carlos, CA, USA, catalogue number S-2108 for CRH). Ranges of the calibrated curve were 20–500 pg/ml for the ACTH assay and 10–1280 pg/ml for the CRH assay. Perfusates for the CRH determination were freeze-dried and then dissolved in 120 ll of RIA buffer to concentrate the samples within the measuring range.
Statistical analysis The significance of differences in ACTH, cortisol and prolactin concentrations among all the experimental groups was examined by one-way ANOVA followed by the least significance differences post-hoc test (STATISTICA; StatSoft, Inc., Tulsa, OK, USA). For nonparametric statistics, the Kruskal–Wallis test followed by multiple comparisons of average ranks for all tests was used to determine the significance of the differences in CRH, salsolinol, noradrenaline and dopamine concentrations among the experimental groups. All data are expressed as the mean SEM.
Results Exposition to stress Both low and high stress evoked a significant (P < 0.001) increase in the concentration of plasma components of the HPA axis (i.e. ACTH and cortisol), whereas no considerable stress-induced changes were observed in the perfusate CRH level. A significant (P < 0.001) increase in noradrenaline concentration was noted in all stressed animals, with the highest value in the High Stress group. Also dopamine concentration increased clearly in both stressed groups, compared to controls, in which dopamine was at an undetectable level. No significant differences were found in salsolinol concentration amongst the Non Stress, Low Stress and High Stress groups. The prolactin concentration even decreased significantly (P < 0.01) in the Low Stress group in comparison with the No Stress group. A clear response to both kinds of stress was also observed in the profiles of ACTH and cortisol secretion, whereas the prolactin profile is similar in control and stressed animals (Figs 1A, 2A and 3A, respectively).
Exposition to low stress and effect of suckling Suckling significantly (P < 0.001) reduced the ACTH and cortisol responses to stress to a similar level as in the No Stress group. No significant changes in CRH level were observed in the Low Stress + Suckling group, compared to the No Stress and Low Stress groups, although, visually, the perfusate CRH concentration in this first was the lowest. Noradrenaline and dopamine concentrations, Journal of Neuroendocrinology, 2014, 26, 844–852
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(A) 250
Fig. 1. Mean plasma adrenocotricotrophic hormone (ACTH) concentrations in consecutive blood samples in accordance with the type of treatment. (A) Exposition to stress (groups: No stress, Low Stress, High Stress). (B) Exposition to low stress and effect of suckling (groups: No Stress, Low Stress, Low Stress + Suckling). (C) Exposition to high stress and effect of salsolinol (groups: No Stress, High Stress, High Stress + Salsolinol). (D) Exposition to low stress and effect of 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ) (No Stress, Low Stress, Low Stress + 1-MeDIQ).
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No Stress and Low Stress groups. The changes in the ACTH, cortisol and prolactin concentrations are clearly reflected by the profiles of secretion monitored during the experiment (Figs 1B, 2B and 3B, respectively).
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Salsolinol infused i.c.v. significantly reduced the ACTH (P < 0.001) and cortisol (P < 0.05) responses to high stress but did not affect the perfusate CRH concentration. However, noradrenaline and dopamine concentrations decreased significantly (P < 0.001) in sheep exposed to high stress, following salsolinol infusion, in comparison with the sheep only stressed. Although, the concentration of salsolinol in the High Stress + Salsolinol group was above the range of analysis, prolactin was even lower than that in the No Stress group. A reduction of the HPA axis response to stress by salsolinol is clearly pronounced in the profile of ACTH secretion (Fig. 1C). Cortisol release returned to the normal rhythm after the first burst following the stress exposition (Fig. 2C).
150 100 50 0 10
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although increased in stressed animals, were not changed statistically by suckling. However, suckling significantly (P < 0.001) increased salsolinol and prolactin concentration compared to the Journal of Neuroendocrinology, 2014, 26, 844–852
1-MeDIQ treatment of the Low Stress group significantly (P < 0.001) raised CRH and cortisol concentration, compared to the No Stress and only Low Stress groups. In case of ACTH, the concentration noted in the Low Stress + 1-MeDIQ group was significantly (P < 0.001) lower than in the Low Stress group but still significantly (P < 0.01) higher than in the No Stress group. 1-MeDIQ infused i.c.v. did not change the noredrenaline, dopamine and prolactin concentrations significantly, compared to the Low Stress group, but reduced the salsolinol level below the assay detection limit. The changes in the ACTH and cortisol concentrations, or their lack as in case of prolactin, are clearly reflected by the profiles of secretion, monitored during the experiment (Figs 1D, 2D and 3D, respectively). The mean concentrations of each studied catecholamine and hormone are shown in Table 2, in accordance with the type of treatments and with a single threshold for statistical significance (P < 0.05).
Discussion The present study extends on existing knowledge concerning the attenuation of the stress response of the HPA axis in lactating sheep and, for the first time, demonstrates the central action of salsolinol in this process. We used two experimental in vivo techniques (i.e. i.c.v. infusion, combined with the push–pull perfusion of © 2014 British Society for Neuroendocrinology
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Cortisol (ng/ml)
(A) 80
Fig. 2. Mean plasma cortisol concentrations in consecutive blood samples collected in accordance with the type of treatment. (A) Exposition to stress (groups: No stress, Low Stress, High Stress); (B) Exposition to low stress and suckling effect (groups: No Stress, Low Stress, Low Stress + Suckling). (C) Exposition to high stress and salsolinol effect (groups: No Stress, High Stress, High Stress + Salsolinol). (D) Exposition to low stress and 1-methyl3,4-dihydro-isoqinoline (1-MeDIQ) effect (No Stress, Low Stress, Low Stress + 1-MeDIQ).
60 40 20 0 10
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the IN/ME) for simultaneous administration of the drug and identification of the substances released from the nerve terminals into the hypophyseal portal system. Besides direct cannulation of the © 2014 British Society for Neuroendocrinology
hypophyseal portal vessels (27), the push–pull perfusion of the IN/ ME is a useful technique, providing information about neural factors, neurotransmitters and neurohormones reaching the anterior pituitary (28,29). Changes in both ACTH and cortisol secretion are the most common plasma measures that indicate the HPA axis response to stress. In the present study, plasma ACTH and cortisol concentrations in lactating sheep increased significantly in response to isolation from the flock, either with lambs present but unable to suckle or when lambs were also isolated. Interestingly, there were no differences in the strength of the stress response between these two groups as a result of similar mean ACTH and cortisol concentrations. This is contrary to the findings of the study by Tilbrook et al. (4) in lactating sheep, which showed that the reaction to isolation and restrain stress was higher in sheep isolated from lambs than in sheep with lambs present but unable to suckle. By contrast to plasma components of the HPA axis, stress did not affect the CRH concentration within the IN/ME, whereas noradrenaline release in this site intensified depending on the strength of the stress. Our data suggest that, during lactation, another hypothalamic factor could be more potent in stimulating ACTH release in response to stress. Because the ACTH response to AVP increased in lactating rats compared to virgins (9), this neuropeptide is the most likely candidate. Moreover, the lack of CRH response to a given type of stress might result from a lesser sensitivity of the PVN to the noradrenergic input during lactation, as described in rats (8). The suckling stimulus is assigned a crucial inhibiting role in the stress-induced activity of the HPA axis during lactation (8) and the present study strongly confirmed this phenomenon. As expected, suckling by the lambs significantly suppressed an increase in both plasma ACTH and cortisol concentration in response to isolation stress. This is consistent with the earlier study, where a greater cortisol response to isolation and restrain stress occurred in sheep that stayed with their lamb unable to suckle than in sheep whose lambs had free access to the udder (4). However, again, there is a discrepancy between the present study and the study by Tilbrook et al. (4) in terms of plasma ACTH concentration because, in the latter study, there were no differences in ACTH concentration between these two groups. We observed a significantly lower plasma concentration of ACTH in stressed sheep suckled by their lamb compared to sheep whose lambs had restricted access to the udder. This might be a result of differences in an experimental set-up such as the period of lactation used or the fact that, in the present study, all lambs had a 1-h limited access to the udder before the actual experiment. The attenuation phenomenon of the stress response of the HPA axis by suckling may partly result from the action of salsolinol. Our Journal of Neuroendocrinology, 2014, 26, 844–852
Suckling and salsolinol attenuates HPA axis activity
Prolactin (ng/ml)
(A) 450 400 350 300 250 200 150 100 50 0
Fig. 3. Mean plasma prolactin concentrations in consecutive blood samples in accordance with the type of treatment. (A) Exposition to stress (groups: No stress, Low Stress, High Stress). (B) Exposition to low stress and suckling effect (groups: No Stress, Low Stress, Low Stress + suckling). (C) Exposition to high stress and salsolinol effect (groups: No Stress, High Stress, High Stress + Salsolinol). (D) Exposition to low stress and 1-methyl-3,4-dihydroisoqinoline (1-MeDIQ) effect (No Stress, Low Stress, Low Stress + 1-MeDIQ).
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earlier studies showed that this dopamine-derived compound is present in the IN/ME of lactating sheep and its concentration increases during suckling (12). This is also confirmed in the present Journal of Neuroendocrinology, 2014, 26, 844–852
study. Moreover, we have shown that salsolinol infused into the IIIv inhibited the stress-induced release of peripheral components of the HPA axis (i.e. ACTH and cortisol), although it had no effect on the CRH concentration. In turn, the use of a structural analogue of salsolinol (i.e. 1-MeDIQ), known to be an inhibitor of some of the actions of salsolinol (21,23), provided some unexpected data. The infusion of 1-MeDIQ into the IIIv caused an exceptional increase in CRH concentration in perfusates collected from the IN/ME, compared to the other experimental groups. Because 1-MeDIQ completely excluded salsolinol from the extracellular matrix of the IN/ ME, our data suggest that salsolinol, indeed, could play a meaningful role in inhibiting CRH release during lactation. This point of view, however, is not reflected by an increase in the mean plasma ACTH concentration of lightly stressed sheep, in which 1-MeDIQ was expected to potentiate the stress response as a whole. However, the same sheep had the highest plasma cortisol concentration, compared to all the other groups, and so a possible explanation could be the strong feedback of cortisol on ACTH in this group. The action of 1-MeDIQ by itself on corticotrophs and adrenal cortex cells cannot be excluded, although it is less probable because the peripheral injection of 1-MeDIQ did not attenuate corticosterone response to immobilisation stress in male rats (21). The detailed central mechanism for the action of salsolinol on the individual components of the HPA axis is not clear. The present study revealed a possible dual effect of this isoquinoline. First, because a meaningful reduction of salsolinol within the IN/ME by 1-MeDIQ caused a more than three-fold increase of CRH concentration, salsolinol appears to be a major inhibiting factor of the stress-induced CRH release in lactating sheep. Second, salsolinol at a higher concentration, following suckling or infusion, reduced the stress-induced noradrenaline release. This neurotransmitter released within the PVN from the brainstem noradrenergic nerve terminals is known to be crucial for activation of the HPA axis by stress (30). During lactation, changes in this system are partly accountable for the reduced HPA axis response to stress (6). The reduced noradrenergic activity is associated with the suckling stimulus because females separated from their pups 48 h earlier have a higher basal noradrenaline concentration in the PVN than suckled females (5). In the present study, we measured the noradrenaline concentration within the extracellular matrix of the IN/ME, in addition to CRH. Thus, the concentration of noradrenaline derived from this site may not fully reflect the amount of this neurotransmitter reaching the PVN but rather the amount delivering to the anterior pituitary. Noradrenaline may also possibly affect the HPA axis acting directly on the pituitary corticotrophs. b-adrenergic receptors were found in the anterior pituitary of several species (31,32) and noradrenergic nerve terminals were located in the ovine infundibular nucleus (33). © 2014 British Society for Neuroendocrinology
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Table 2. The Mean Concentrations of Catecholamines (Noradrenaline, Dopamine, Salsolinol) and Hormones [Corticotrophin-Releasing Hormone (CRH), Adenocorticotrophic Hormone (ACTH), Cortisol, Prolactin] in Accordance with the Type of Treatment: Exposition to Stress (Groups: No Stress, Low Stress, High Stress); Exposition to Low Stress and Effect of Suckling (Groups: No Stress, Low Stress, Low Stress + Suckling); Exposition to High Stress and Effect of Salsolinol (Groups: No Stress, High Stress, High Stress + Salsolinol); and Exposition to Low Stress and Effect of 1-methyl-3,4-dihydro-isoqinoline (1-MeDIQ) (Groups: No Stress, Low Stress, Low Stress + 1-MeDIQ). HPA axis
Group
CRH (pg/ml)
Catecholamines ACTH (pg/ml)
Exposition to stress No stress 35.61 4.30 59.73 Low stress 40.82 3.38 97.19 High stress 42.22 6.79 101.13 Exposition to low stress and effect of suckling No stress 35.61 4.30 59.73 Low stress 40.82 3.38 97.19 Low stress + suckling 32.04 3.29 50.90 Exposition to high stress and effect of salsolinol No stress 35.61 4.30 59.73 High stress 42.22 6.79 101.13 High stress + salsolinol 48.49 5.54 58.79 Exposition to low stress and effect of 1-MeDIQ 59.73 No stress 35.61 4.30a Low stress 40.82 3.38a 97.19 Low stress + 1-MeDIQ 134.74 15.26b 74.94
Cortisol (ng/ml)
Noradrenaline (pg/50 ll)
Dopamine (pg/50 ll)
Salsolinol (pg/50 ll)
Prolactin (ng/ml)
2.48a 5.09b 6.80b
18.52 1.23a 29.20 1.56b 26.21 1.61b
41.71 2.51a 89.97 6.72b 139.49 7.00c
Below detection 96.59 16.17 114.65 14.25
23.78 4.48 32.39 6.10 32.44 3.88
110.79 8.45a 77.79 7.81b 89.23 5.08
2.48a 5.09b 1.75a
18.52 1.23a 29.20 1.56b 14.03 0.87a
41.71 2.51a 89.97 6.72b 66.78 5.68b
Below detection 96.59 16.17 50.81 5.27
23.78 4.48a 32.39 6.10a 227.49 11.47b
110.79 8.45a 77.79 7.81b 276.16 14.18c
2.48a 6.80b 1.82a
18.52 1.23a 26.21 1.61b 20.55 1.09a
41.71 2.51a 139.49 7.00b 57.22 4.78a
Below detection 114.65 14.25b 44.11 10.19a
23.78 4.48 32.44 3.88 Above detection
110.79 8.45a 89.23 5.08 73.02 2.81b
2.48a 5.09c 2.74b
18.52 1.23a 29.20 1.56b 38.20 2.60c
41.71 2.51a 89.97 6.72b 80.91 7.04b
Below detection 96.59 16.17 65.28 6.53
23.78 4.48 32.39 6.10 Below detection
110.79 8.45a 77.79 7.81b 93.18 4.93
HPA, hypothalamic-pituitary-adrenal. Different superscript lowercase letters indicate a statistically significant difference (P < 0.05).
Moreover, noradrenaline stimulated the release of ACTH from cultured ovine anterior pituitary cells (34). Regardless of the pathway for the action of salsolinol, the inhibitory mechanism for stressinduced activity of the HPA axis in nursing females could also be a consequence of reduced noradrenaline release. This is in line with the latest research showing that salsolinol inhibits tyrosine hydroxylase (the rate-limiting enzyme of the catecholamines biosynthesis) activity (35). Because salsolinol is known as a prolactin-releasing factor (36) and prolactin receptors are present in the hypothalamus and other regions of the central nervous system (37), this hormone appears to mediate the discussed phenomenon of the action of salsolinol on the HPA axis. In the present study, stress was an inefficient stimulus for increasing the plasma prolactin concentration in contrast to suckling. It might be a result of stress-induced dopamine release within the IN/ME, which could prevent an additional increase in stress-induced prolactin. Interestingly, although the infusion of salsolinol caused an inhibition of ACTH-cortisol response to high stress, this compound was unable to trigger the release of extra prolactin into circulation. These data suggest that salsolinol is ineffective as a prolactin-releasing factor in lactating sheep during stressful conditions and it is unlikely that prolactin mediates the inhibitory effect of salsolinol on the stress-induced activity of the HPA axis. Therefore, another factor(s) should be taken into account in suckling stimulation of prolactin release under these unfavourable conditions and oxytocin may be one of the candidates. © 2014 British Society for Neuroendocrinology
Evidence exists suggesting that oxytocin mediates the prolactin surge induced by suckling (38) and, moreover, is able to suppress the cortisol response to an audiovisual stressor in nonlactating and lactating sheep (39). This, however, is beyond the scope of the present study and requires further investigation. In summary, our research points to the profound changes with respect to regulating the stress response that occurs during lactation and the relevant role of salsolinol at this physiological stage. The reported results suggest that salsolinol mediates the inhibiting effect of suckling on stress-induced HPA axis activity in lactating sheep. A better understanding of the molecular action of salsolinol is necessary to confirm this and explain precisely the physiological actions of this compound.
Acknowledgements This research was supported by the National Science Centre, Poland, Grant number N N311 082037. The authors would like to thank Professor F. Fulop (Szeged University, Hungary) for preparing and providing salsolinol and 1-MeDIQ; veterinary surgeon J. Rutkowski for help with the brain surgery; K. Romanowicz PhD for help with the hormone RIA; K. Gorski PhD and E. Marciniak MSc for help with blood sampling; and Mr W. Mrozek for animal care. The authors have no conflicts of interest to declare.
Received 22 November 2013, revised 12 April 2014, accepted 30 August 2014 Journal of Neuroendocrinology, 2014, 26, 844–852
Suckling and salsolinol attenuates HPA axis activity
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