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Possible role of adrenoceptors in the hypothalamic paraventricular nucleus in corticotropin-releasing factor-induced sympatho-adrenomedullary outflow in rats
Autonomic Neuroscience: Basic and Clinical 203 (2017) 74–80
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Possible role of adrenoceptors in the hypothalamic paraventricular nucleus in corticotropin-releasing factor-induced sympatho-adrenomedullary outflow in rats Shoshiro Okada ⁎, Naoko Yamaguchi Department of Pharmacology, Graduate School of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
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Article history: Received 7 September 2016 Received in revised form 30 December 2016 Accepted 20 January 2017 Keywords: Adrenoceptor Corticotropin-releasing factor Noradrenaline release Paraventricular nucleus of the hypothalamus Plasma catecholamines
a b s t r a c t Aims: A functional interaction between the corticotropin-releasing factor (CRF) system and noradrenergic neurons in the brain has been suggested. In the present study, we investigated the interrelationship between the central CRF-induced elevation of plasma catecholamines and adrenoceptor activation in the paraventricular nucleus of the hypothalamus (PVN) using urethane-anesthetized rats. Main methods: In rats under urethane anesthesia, a femoral venous line was inserted for infusion of saline, and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for the application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed with high-performance liquid chromatography with electrochemical detection. Quantification of noradrenaline in rat PVN microdialysates was performed with high-performance liquid chromatography with electrochemical detection. Key findings: We showed that centrally administered CRF elevated noradrenaline release in the PVN. Furthermore, we demonstrated that microinjection of phenylephrine into the PVN induced elevation of plasma levels of adrenaline, but not of noradrenaline, whereas microinjection of isoproterenol into the PVN induced elevation of plasma levels of noradrenaline, but not of adrenaline. Bilateral blockade of adrenoceptors in the PVN revealed that phentolamine significantly suppressed the CRF-induced elevation of plasma adrenaline level, while propranolol significantly CRF-induced elevation of plasma noradrenaline level. Significance: Our results suggest that centrally administered CRF-induced elevation of plasma levels of adrenaline and noradrenaline can be mediated via activation of α-adrenoceptors and β-adrenoceptors, respectively, in the rat PVN. © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Corticotropin-releasing factor (CRF), a 41-amino acid peptide, is a key stress-related neuropeptide and was originally established as the primary physiological regulator of adrenocorticotropic hormone (ACTH) secretion (Rivier and Vale, 1985). However, it has been well documented that the peptide exhibits a broad distribution in extrahypothalamic brain regions, acting as a neurotransmitter/ neuromodulator, and forms circuits in the brain (Sawchenko and Swanson, 1983; Cummings and Seybold, 1988). Increasing evidence indicates that within the central nervous system, the peptide also acts as a regulator of the autonomic nervous system and of cardiovascular function (Brown and Fisher, 1985; Brown et al., 1985; Fisher, 1988; Yokotani et al., 2001; Okada et al., 2003). Specifically, Brown et al. (1982) ⁎ Corresponding author at: Department of Pharmacology, Graduate School of Medicine, Aichi Medical University, Japan. E-mail address: [email protected] (S. Okada).
reported that intracerebroventricularly administered CRF produces an elevation of the plasma concentrations of noradrenaline and adrenaline in conscious rats. They revealed that CRF acts within the brain to stimulate sympathetic outflow by experiments with hypophysectomy, adrenalectomy and ganglion blocker, indicating that the central effects of CRF is thought to be entirely independent of ACTH release. By using selective receptor antagonists, we previously reported that the centrally administered CRF-induced elevation of plasma noradrenaline was mediated by the activation of α1 and β adrenoceptors in the brain, and that of plasma adrenaline is mediated by the activation of α1 adrenoceptors in the brain (Yorimitsu et al., 2008). More recently, we reported that intracerebroventricularly administered isoproterenol, a β adrenoceptor agonist, elevated plasma noradrenaline, but not adrenaline (Ando et al., 2015). Collectively, these observations suggest that centrally administered CRF might elevate plasma catecholamines via activation of brain adrenoceptors. Noradrenaline is one of the major neurotransmitters involved in brain function, and it acts as a warning signal under stress conditions.
http://dx.doi.org/10.1016/j.autneu.2017.01.008 1566-0702/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Okada, N. YamaguchiAutonomic Neuroscience: Basic and Clinical 203 (2017) 74–80
It activates α or β adrenoceptors in the brain to elicit several kinds of biological responses, such as increased blood pressure and heart rate and secretion of ACTH (Moore and Bloom, 1979; Woodruff et al., 1986; McCall, 1988; Schreihofer and Guyenet, 2000). The paraventricular nucleus of the hypothalamus (PVN) is a complex integrative center important for neurohumoral regulation and the maintenance of cardiovascular and body fluid homeostasis (Kenney et al., 2003; Benarroch, 2005). The PVN receives dense noradrenergic projections from the brainstem with an additional smaller contribution arising from the locus coeruleus (Sawchenko and Swanson, 1983). An ultrastructural study demonstrated that noradrenergic axon terminals contact gastric pre-autonomic neurons in the PVN (Balcita-Pedicino and Rinaman, 2007). Furthermore, functional analyses suggest that adrenoceptors in the PVN play an important role in regulating sympathetic outflow (Scheurink et al., 1990; Williams and Morilak, 1997; Chen et al., 2006; Zhang and Felder, 2008). Collectively, these observations suggest that noradrenergic neurons projecting to the PVN can activate adrenoceptors located in the PVN and can mediate the excitatory effects of noradrenaline to induce sympathetic outflow. In the present study, to identify the brain sites for CRF-induced sympatho-adrenomedullary outflow, we used a brain microdialysis technique to examine pharmacologically the possibility that centrally administered CRF can release noradrenaline in the PVN. Furthermore, the effects of microinjection into the PVN of phenylephrine, an α1adrenoceptor agonist, or isoproterenol, a β adrenoceptor agonist on the plasma levels of noradrenaline and adrenaline were examined. 2. Materials and methods 2.1. Animals Male Wistar rats weighing approximately 350 g were maintained in an air-conditioned room at 22–24 °C under a constant day-night rhythm for N2 weeks and given food (laboratory chow, CE-2; CLEA Japan, Hamamatsu, Japan) and water ad libitum. All experiments were conducted in compliance with the guiding principles for the care and use of laboratory animals approved by Aichi Medical University. 2.2. Microdialysis Microdialysis experiments were performed according to methods published in a previous report (Okada et al., 2000). Briefly, a stainless steel guide cannula held on the tip of an L-shaped stainless steel apparatus was implanted stereotaxically just above the right PVN. For intracerebroventricular administration of CRF, another stainless steel cannula (o.d. 0.35 mm) was inserted into the left lateral cerebral ventricle, and the peptide was applied in a volume of 10 μl using a 50-μl Hamilton syringe at the following coordinates (in mm): AP +1.59, L 1.5, V − 4.62 (AP, anterior from bregma; L, lateral from the midline; V, below the surface of the brain). The PVN was perfused with Ringer's solution (147 mM NaCl, 4 mM KCl and 2.3 mM CaCl2) at a flow rate of 2 μl/ min using a microinfusion pump (EP-60, Eicom, Kyoto, Japan). Three consecutive dialysates were used to measure the baseline release of noradrenaline. Noradrenaline was measured directly with high-performance liquid chromatography (HPLC) with electrochemical detection (Okada et al., 2002).
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− 1.8 mm; L, 0.3 mm; V, 7.6 mm: lateral ventricle: AP, − 0.8 mm; L, 1.5 mm; V, 4.0 mm, according to the rat brain atlas of Paxinos and Watson (2005). Four hours were allowed to elapse before the application of test substances. In the experimental group, a stainless steel injection cannula (o.d. 0.3 mm) was attached to PE-20 tubing, which was then connected to a Hamilton microsyringe (0.5-μl syringe for PVN injection; 10-μl syringe for intracerebroventricular injection). To examine effects of adrenoceptor agonists on plasma catecholamine levels, phenylephrine (5 or 10 nmol), isoproterenol (5 or 10 nmol) or sterile saline as a vehicle was injected unilaterally into the PVN in a volume of 50 nl over 30 s. Next, to examine effects of bilateral blockade of adrenoceptors in the PVN on CRF-induced elevation of plasma catecholamine levels, adrenoceptor antagonists in combination with CRF were administered. Phentolamine (6 nmol/side), propranolol (6 nmol/side) or dimethyl sulfoxide (DMSO) as a vehicle was injected bilaterally into the PVN in a volume of 100 nl over 60 s. After 30 min, CRF (1.5 nmol) or sterile saline was injected intracerebroventricularly in a volume of 10 μl over 30 s. At the end of the experiments, brains were removed, post-fixed in 4% paraformaldehyde solution and cut on a cryostat, and then brain sections were stained in hematoxylin solution for histological verification of injection sites. 2.4. Measurement of plasma catecholamines Arterial blood samples (250 μl) were collected in a heparinized tube through an arterial catheter and were preserved on ice during the experiments. Immediately after the final sampling, plasma was prepared by centrifugation (3000 ×g for 10 min at 4 °C). According our previous reports, catecholamines in the plasma were extracted by alumina and were assayed with HPLC with electrochemical detection (Ando et al., 2015). 2.5. Treatment of data and statistics Results are expressed as the mean ± S.E.M. of the net change above the respective basal value. The data were analyzed by Student's t-test (Fig. 1) or by one-way ANOVA with SPSS v22.0, followed by a posthoc analysis with the Bonferroni method (Figs. 2, 3, 5). P values b 0.05 were taken to indicate statistical significance.
2.3. Microinjection With rats under urethane anesthesia (1.2 g/kg, intraperitoneally), a femoral venous line was inserted for infusion of saline (1.2 ml/h), and an arterial line was inserted for collection of blood samples, as described previously (Ando et al., 2015). Next, the animal was placed in a stereotaxic apparatus, as described previously (Ando et al., 2015). Holes were drilled in the skull for administration of test substances. The stereotaxic coordinates of the tip of the cannula were as follows (in mm): PVN: AP,
Fig. 1. Effects of intracerebroventricularly (i.c.v.) administered CRF on the release of noradrenaline in the PVN. ●, CRF (n = 6); ○, vehicle (10 μl saline/animal, i.c.v.) (n = 4). *Significantly different (pb0.05) from vehicle.
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Fig. 2. Effects of PVN microinjection of the α1-adrenergic agonist phenylephrine on plasma levels of noradrenaline and adrenaline. Arrows indicate each PVN microinjection. Phenylephrine (5 and 10 nmol/animal) or vehicle (saline) was administered in a volume of 50 nl. ○, vehicle (saline) (n = 6); ●, phenylephrine (5 nmol/animal) (n = 6); ▲, phenylephrine (10 nmol/animal) (n = 6). *Significantly different (p b 0.05) from vehicle-treated control. Each point represents the mean ± S.E.M.
2.6. Drugs The following pharmacological agents were used: synthetic corticotropin-releasing hormone (rat/human) (Peptide Institute, Osaka, Japan); phenylephrine, isoproterenol and phentolamine (Sigma-Aldrich Chemicals, St. Louis, MO); propranolol (Wako Pure Chemical Industries, Osaka, Japan). All other reagents were the highest grade available (Nacalai Tesque, Kyoto, Japan).
3. Results 3.1. Effect of CRF on noradrenaline release in the PVN The mean baseline level of noradrenaline in the microdialysate (three consecutive samples) was 0.9 ± 0.1 pg/20 min-fraction (n = 70). Application of Ringer's solution into the PVN with a microdialysis probe did not alter the baseline release of noradrenaline in the PVN (Fig. 1). Application of CRF (1.5 nmol/animal) into the left ventricle elevated the release of noradrenaline in the PVN. After the application of CRF, the maximal response of noradrenaline release was observed at 80 min, and then it declined to the baseline level (Fig. 1).
3.2. Effects of phenylephrine, an α1-adrenoceptor agonist, on plasma catecholamine levels after microinjection into the PVN The 50 nl volume of saline microinjected into the PVN and blood sampling six times over a 60-min period had no significant effect on the baseline plasma levels of either noradrenaline or adrenaline (Fig. 2). Unilateral microinjection of phenylephrine (5 and 10 nmol/50 nl) directly into the PVN dose-dependently elevated the plasma levels of adrenaline, while it did not alter the plasma levels of noradrenaline (Fig. 2). The adrenaline levels peaked at 10 min and then gradually declined toward baseline. The actual values for noradrenaline and adrenaline at 0 min were 303.8 ± 67.9 and 394.4 ± 85.3 pg/ml, respectively, for the saline-treated group (n = 6), 493.6 ± 125.5 and 339.7 ± 44.2 pg/ml, respectively, for the 5 nmol phenylephrine-treated group (n = 6), and 338.7 ± 80.0 and 581.5 ± 133.4 pg/ml, respectively, for the 10 nmol phenylephrine-treated group (n = 6).
3.3. Effects of isoproterenol, a β-adrenoceptor agonist, on plasma catecholamine levels after microinjection into the PVN The 50 nl volume of saline microinjected into the PVN and blood sampling six times over a 60-min period had no significant effect on
Fig. 3. Effects of PVN microinjection of the β-adrenergic agonist isoproterenol on plasma levels of noradrenaline and adrenaline. Arrows indicate each PVN microinjection. Isoproterenol (5 and 10 nmol/animal) or vehicle (saline) was administered in a volume of 50 nl. ○, vehicle (saline) (n = 5); ●, isoproterenol (5 nmol/animal) (n = 7); ▲, isoproterenol (10 nmol/animal) (n = 8). *Significantly different (p b 0.05) from vehicle-treated control. Each point represents the mean ± S.E.M.
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the baseline plasma levels of either noradrenaline or adrenaline (Fig. 3). Unilateral microinjection of isoproterenol (10 nmol/50 nl) directly into the PVN significantly elevated the plasma levels of noradrenaline (Fig. 3). The noradrenaline levels peaked at 5 min and then gradually declined toward baseline. In contrast, only the highest dose of isoproterenol (10 nmol/animal) significantly elevated the plasma levels of adrenaline (Fig. 3). The actual values for noradrenaline and adrenaline at 0 min were 384.4 ± 38.9 and 263.8 ± 81.1 pg/ml, respectively, for the saline-treated group (n = 5), 271.9 ± 51.6 and 215.4 ± 72.8 pg/ml, respectively, for the 5 nmol isoproterenol-treated group (n = 7), and 351.9 ± 21.1 and 117.1 ± 31.4 pg/ml, respectively, for the 10 nmol isoproterenol-treated group (n = 8). 3.4. Histological verification of unilateral microinjection sites in the PVN A typical unilateral microinjection site in the PVN is shown in Fig. 4A. Diagrams showing microinjection sites in the PVN at different
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rostrocaudal levels (1.56 to 1.92 mm caudal to bregma) are depicted in Fig. 4B. In these diagrams, each symbol represents a microinjection site in one animal. Data from rats for which microinjection sites were confirmed were used for analysis. 3.5. Effects of bilateral microinjections of phentolamine, an α-adrenoceptor antagonist, or propranolol, a β-adrenoceptor antagonist, into the PVN on the CRF-induced elevation of plasma catecholamine levels The bilateral microinjections of vehicle-1 (100 nl of DMSO) into the PVN in combination with vehicle-2 (10 μl of saline) injected into the lateral ventricle had no significant effect on the plasma levels of catecholamines (Fig. 5). The bilateral microinjections of vehicle-1 (100 nl of DMSO) into the PVN in combination with CRF (1.5 nmol/10 μl) injected into the lateral ventricle had significantly elevated plasma catecholamine levels (Fig. 5). The bilateral microinjections of phentolamine (6 nmol/100 nl/side) into the PVN did not affect the CRF-induced
Fig. 4. Microinjection sites in the PVN. (A) Photomicrograph of a coronal rat brain section indicating the injection sites in the PVN. Scale bar = 200 μm. (B) Diagrammatic representation of rat brain frontal sections, from Paxinos and Watson (2005), showing the distribution of injection sites in phenylephrine (black) and isoproterenol (white) in the unilateral PVN of urethane-anesthetized rats (circle, vehicle; triangle, 5 nmol; square, 10 nmol). 3V, third ventricle; AH, anterior hypothalamic area; f, fornix; PVN, paraventricular nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.
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Fig. 5. Effects of bilateral microinjections of phentolamine, an α-adrenoceptor antagonist, or propranolol, a β-adrenoceptor antagonist into the PVN on the CRF-induced elevation of plasma catecholamine levels. Arrows indicate each PVN microinjection and intracerebroventricular administration. Phentolamine (6 nmol/side), propranolol (6 nmol/side) or vehicle-1 (DMSO) was microinjected bilaterally into the PVN in a volume of 100 nl. CRF (1.5 nmol/animal) or vehicle-2 (saline) was administered intracerebroventricularly in a volume of 10 μl. ○, vehicle1 + CRF (n = 6); ■, phentolamine + CRF (n = 5); ▲, propranolol + CRF (n = 5); ●, vehicle-1 + vehicle-2 (n-5). *Significantly different (p b 0.05) from vehicle-1 + CRF-treated group. Each point represents the mean ± S.E.M.
elevation of plasma noradrenaline level, but suppressed significantly the CRF-induced elevation of plasma adrenaline level (Fig. 5). In contrast, microinjections of propranolol (6 nmol/100 nl/side) into the PVN significantly suppressed the CRF-induced elevation of plasma noradrenaline level, while those of propranolol showed no statistically significant change on the CRF-induced elevation of plasma adrenaline level, although those of propranolol tended to suppress the elevation of plasma adrenaline level at 60 and 90 min (p = 0.092 at 60 min; p = 0.056 at 90 min). The actual values for noradrenaline and adrenaline at 0 min were 204.8 ± 65.6 and 151.9 ± 57.5 pg/ml for vehicle-1 plus vehicle-2-treated group (n = 5), 343.8 ± 40.4 and 210.8 ± 30.5 pg/ml for vehicle-1 plus CRF-treated group (n = 6), 346.8 ± 104.2 and 400.3 ± 124.1 pg/ml for phentolamine plus CRF-treated group (n = 5), and 289.4 ± 41.5 and 127.5 ± 55.2 pg/ml for propranolol plus CRF-treated group (n = 5), respectively. 3.6. Histological verification of bilateral microinjection sites in the PVN A typical bilateral microinjection site in the PVN is shown in Fig. 6A. Diagrams showing microinjection sites in the PVN at different rostrocaudal levels (1.56 to 1.92 mm caudal to bregma) are depicted in Fig. 6B. In these diagrams, each symbol represents a microinjection site in one animal. Data from rats for which microinjection sites were confirmed were used for analysis. 4. Discussion It has been shown that central administration of CRF induces autonomic activation, resulting in increases in blood pressure, heart rate and plasma catecholamines (Brown et al., 1982; Korte et al., 1993; Yokotani et al., 2001; Okada et al., 2003). Immunohistochemical analyses revealed that intracerebroventricularly administered CRF evokes Fos expression in the PVN, a key center for central autonomic control (Bittencourt and Sawchenko, 2000). Furthermore, several lines of evidence indicate that centrally administered CRF activates noradrenergic neurons projecting to the PVN and induces an increase in noradrenaline release in the PVN (Otagiri et al., 2000; Dunn et al., 2004; Okada et al., 2008). Collectively, these observations may imply a functional interaction between central CRF and noradrenergic neurons projecting to the PVN. However, the precise mechanisms underlying these actions of CRF are still not fully defined.
In the present study, we showed that centrally administered CRF increased noradrenaline release in the PVN in urethane-anesthetized rats, consistent with previous reports using different experimental conditions (Dunn et al., 2004; Okada et al., 2008). Thus, it is conceivable that increased noradrenaline released in the PVN might stimulate adrenoceptors located in the PVN. Since there is considerable evidence that adrenoceptors are abundant in the PVN (Scriabine et al., 1976; McCall and Humphrey, 1981), we next performed microinjection experiments with α and β adrenoceptor agonists. Interestingly, α-adrenoceptor activation by phenylephrine increased plasma levels of adrenaline, whereas βadrenoceptor activation by isoproterenol increased plasma levels of noradrenaline. Therefore, their physiological roles must be different. It has been reported that bilateral microinjection of phenylephrine directly into the PVN increases renal sympathetic activity and heart rate, but it did not affect mean arterial pressure (Zhang and Felder, 2008). More recently, Yamaguchi (2013) demonstrated that administration of isoproterenol into the PVN increased the heart rate in freely moving rats. Collectively, these lines of evidence indicate that stimulation of α or β-adrenoceptor located in the PVN can induce sympathetic outflow. The present study clearly demonstrated that activation of α1adrenoceptors and β-adrenoceptors in the PVN could separately regulate plasma levels of catecholamines in urethane-anesthetized rats. Finally, to reveal whether adrenoceptors located in the PVN mediate the central action of CRF to increase plasma catecholamine, we next examined the effects of α or β adrenoceptor antagonists microinjected into the PVN on the CRF-induced elevations of plasma levels of catecholamine. The present results clearly demonstrated that central CRF-induced elevation of noradrenaline was mediated via activation of β adrenoceptor in the PVN, while that of adrenaline was mediated via activation of α adrenoceptor in the PVN. Thus, it would be reasonable to assume that the endogenous noradrenaline released by CRF stimulation binds to adrenoceptors in the PVN, and that α or β adrenoceptor in the PVN could play differential roles for CRF-induced elevations of plasma catecholamines. Although Itoi et al. (1994) showed that microinjection of noradrenaline into the PVN stimulates CRF secretion into the portal circulation to promote hypothalamic-adrenal axis via activation of α1-adrenergic receptors but not of β adrenergic receptors, there has been no detailed studies regarding roles for adrenoceptors in the PVN responsible for central action of CRF-induced elevation of plasma catecholamine. In light of the present study, one can assume that activation of noradrenergic neurons projecting to the PVN might promote not only
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Fig. 6. Bilateral microinjection sites in the PVN. (A) Photomicrograph of a coronal rat brain section indicating the injection sites in the PVN. Scale bar = 200 μm. (B) Diagrammatic representation of rat brain frontal sections, from Paxinos and Watson (2005), showing the distribution of injection sites. ○, vehicle-1 + CRF; ■, phentolamine + CRF; ▲, propranolol + CRF; ●, vehicle-1 + vehicle-2. 3V, third ventricle; AH, anterior hypothalamic area; f, fornix; PVN, paraventricular nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.
hypothalamic-adrenal axis but also sympatho-adrenomedullary outflow via α or β adrenoceptors. Taking physiological roles for CRF as a stress related peptide into account, the present results may give an opportunity to reveal the detailed mechanisms underlying CRF-induced stress responses. Previously, we reported that central CRF differentially elevated plasma levels of catecholamines, namely, noradrenaline via brain prostaglandin E2 (PGE2) and adrenaline via brain thromboxane A2 (Okada et al., 2003). Recently, a microinjection study revealed that EP3 receptors mediate the central excitatory effects of PGE2 on PVN neurons and sympathetic discharge, such as renal nerve activity, mean blood pressure and heart rate (Zhang et al., 2011). More recently, we reported that centrally administered isoproterenol increased plasma levels of noradrenaline, but not of adrenaline, via PGE2-mediated mechanisms (Ando et al., 2015). These observations raised the possible connections between adrenoceptor activation and mode of action of prostanoids in the PVN. Further analysis of the detailed mechanisms underlying these interactions will be carried out in future studies.
Our previous study demonstrated that CRF-induced sympathoadrenomedullary outflow is mediated by central CRF1 receptors (Yokotani et al., 2001). Although it has been shown that one of the first anatomical sites exposed to centrally-administered CRF is the lateral septum mediolateral nucleus (Liu et al., 2004; Gallagher et al., 2008), the precise brain sites responsible for the various actions of CRF are still unknown. Interestingly, it has been demonstrated that expression of CRF1 receptor mRNA in the PVN is localized principally to autonomic-related aspects of the parvocellular division (Bittencourt and Sawchenko, 2000; Van Pett et al., 2000). In addition, recent reports suggest a possible involvement of presynaptic CRF1 receptors in modulating neurotransmitter release (Gallagher et al., 2008; Kirby et al., 2008; Nie et al., 2009). Collectively, these observations raise an interesting possibility that CRF1 receptors may exist and act on noradrenergic terminals projecting to the PVN to regulate noradrenaline release. In conclusion, we showed that centrally administered CRF elevated noradrenaline release in the PVN. The increased noradrenaline in the PVN might activate α1-adrenoceptors and β-adrenoceptors located in
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the PVN and elevate plasma levels of noradrenaline and adrenaline in urethane-anesthetized rats. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 20591370 to S.O.) from the Japan Society for the Promotion of Science and a Grant-in-Aid for Young Scientists (B) (No. 21790628 to N.Y.). References Ando, K., Kondo, F., Yamaguchi, N., Tachi, M., Fukayama, M., Yoshikawa, K., Gosho, M., Fujiwara, Y., Okada, S., 2015. Centrally administered isoproterenol induces sympathetic outflow via brain prostaglandin E2-mediated mechanisms in rats. Auton. Neurosci. 189, 1–7. Balcita-Pedicino, J.J., Rinaman, L., 2007. Noradrenergic axon terminals contact gastric preautonomic neurons in the paraventricular nucleus of the hypothalamus in rats. J. Comp. Neurol. 501, 608–618. Benarroch, E.E., 2005. Paraventricular nucleus, stress response, and cardiovascular disease. Clin. Auton. Res. 15, 254–263. Bittencourt, J.C., Sawchenko, P.E., 2000. Do centrally administered neuropeptides access cognate receptors?: An analysis in the central corticotropin-releasing factor system. J. Neurosci. 20, 1142–1156. Brown, M.R., Fisher, L.A., 1985. Corticotropin-releasing factor: effects on the autonomic nervous system and visceral systems. Fed. Proc. 44, 243–248. Brown, M.R., Fisher, L.A., Spiess, J., Rivier, C., Rivier, J., Vale, W., 1982. Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology 111, 928–931. Brown, M.R., Fisher, L.A., Webb, V., Vale, W.W., Rivier, J.E., 1985. Corticotropin-releasing factor: a physiologic regulator of adrenal epinephrine secretion. Brain Res. 328, 355–357. Chen, Q., Li, D.P., Pan, H.L., 2006. Presynaptic α1 adrenergic receptors differentially regulate synaptic glutamate and GABA release to hypothalamic presympathetic neurons. J. Pharmacol. Exp. Ther. 316, 733–742. Cummings, S., Seybold, V., 1988. Relationship of alpha-1- and alpha-2-adrenergic-binding sites to regions of the paraventricular nucleus of the hypothalamus containing corticotropin-releasing factor and vasopressin neurons. Neuroendocrinology 47, 523–532. Dunn, A.J., Swiergiel, A.H., Palamarchouk, V., 2004. Brain circuits involved in corticotropin-releasing factor-norepinephrine interactions during stress. Ann. N. Y. Acad. Sci. 1018, 25–34. Fisher, L.A., 1988. Corticotropin-releasing factor: central nervous system effects on baroreflex control of heart rate. Life Sci. 42, 2645–2649. Gallagher, J.P., Orozco-Cabal, L.F., Liu, J., Shinnick-Gallagher, P., 2008. Synaptic physiology of central CRH system. Eur. J. Pharmacol. 583, 215–225. Itoi, K., Suda, T., Tozawa, F., Dobashi, I., Ohmori, N., Sakai, Y., Abe, K., Demura, H., 1994. Microinjection of norepinephrine into the paraventricular nucleus of the hypothalamus stimulates corticotropin-releasing factor gene expression in conscious rats. Endocrinology 135, 2177–2182. Kenney, M.J., Weiss, M.L., Haywood, J.R., 2003. The paraventricular nucleus: an important component of the central neurocircuitry regulating sympathetic nerve outflow. Acta Physiol. Scand. 177, 7–15. Kirby, L.G., Freeman-Daniels, E., Lemos, J.C., Nunan, J.D., Lamy, C., Akanwa, A., Beck, S.G., 2008. Corticotropin-releasing factor increases GABA synaptic activity and induces inward current in 5-hydroxytryptamine dorsal raphe neurons. J. Neurosci. 28, 12927–12937. Korte, S.M., Bouws, G.A., Bohus, B., 1993. Central actions of corticotropin-releasing hormone (CRH) on behavioral, neuroendocrine, and cardiovascular regulation: brain corticoid receptor involvement. Horm. Behav. 27, 167–183. Liu, J., Yu, B., Neugebauer, V., Grigoriadis, D.E., Rivier, J., Vale, W.W., Shinnick-Gallagher, P., Gallagher, J.P., 2004. Corticotropin-releasing factor and Urocortin I modulate excitatory glutamatergic synaptic transmission. J. Neurosci. 24, 4020–4029.
McCall, R.B., 1988. Effects of putative neurotransmitters on sympathetic preganglionic neurons. Annu. Rev. Physiol. 50, 553–564. McCall, R.B., Humphrey, S.J., 1981. Evidence for a central depressor action of postsynaptic alpha 1-adrenergic receptor antagonists. J. Auton. Nerv. Syst. 3, 9–23. Moore, R.Y., Bloom, F.E., 1979. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2, 113–168. Nie, Z., Zorrilla, E.P., Madamba, S.G., Rice, K.C., Roberto, M., Siggins, G.R., 2009. Presynaptic CRF1 receptors mediate the ethanol enhancement of GABAergic transmission in the mouse central amygdala. Sci. World J. 9, 68–85. Okada, S., Murakami, Y., Nishihara, M., Yokotani, K., Osumi, Y., 2000. Perfusion of the hypothalamic paraventricular nucleus with N-methyl-D-aspartate produces thromboxane A2 and centrally activates adrenomedullary outflow in rats. Neuroscience 96, 585–590. Okada, S., Murakami, Y., Yokotani, K., 2002. Centrally applied nitric oxide donor elevates plasma corticosterone by activation of the hypothalamic noradrenergic neurons in rats. Brain Res. 939, 26–33. Okada, S., Murakami, Y., Yokotani, K., 2003. Role of brain thromboxane A2 in the release of noradrenaline and adrenaline from adrenal medulla in rats. Eur. J. Pharmacol. 467, 125–131. Okada, S., Yamaguchi-Shima, N., Shimizu, T., Arai, J., Lianyi, L., Wakiguchi, H., Yokotani, K., 2008. Role of brain nicotinic acetylcholine receptor in centrally administered corticotropin-releasing factor-induced elevation of plasma corticosterone in rats. Eur. J. Pharmacol. 587, 322–329. Otagiri, A., Wakabayashi, I., Shibasaki, T., 2000. Selective corticotropin-releasing factor type 1 receptor antagonist blocks conditioned fear-induced release of noradrenaline in the hypothalamic paraventricular nucleus of rats. J. Neuroendocrinol. 12, 1022–1026. Paxinos, G., Watson, C., 2005. The Rat Brain in Stereotaxic Coordinates. fifth ed. Academic Press, New York. Rivier, C., Vale, W., 1985. Effects of corticotropin-releasing factor, neurohypophyseal peptides, and catecholamines on pituitary function. Fed. Proc. 44, 189–195. Sawchenko, P.E., Swanson, L.W., 1983. The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J. Comp. Neurol. 218, 121–144. Scheurink, A.J., Steffens, A.B., Gaykema, R.P., 1990. Paraventricular hypothalamic adrenoceptors and energy metabolism in exercising rats. Am. J. Physiol 259, R478–R484. Schreihofer, A.M., Guyenet, P.G., 2000. Role of presympathetic C1 neurons in the sympatholytic and hypotensive effects of clonidine in rats. Am. J. Physiol 279, R1753–R1762. Scriabine, A., Clineschmidt, B.V., Sweet, C.S., 1976. Central noradrenergic control of blood pressure. Annu. Rev. Pharmacol. Toxicol. 16, 113–123. Van Pett, K., Viau, V., Bittencourt, J.C., Chan, R.K., Li, H.Y., Arias, C., Prins, G.S., Perrin, M., Vale, W., Sawchenko, P.E., 2000. Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. J. Comp. Neurol. 428, 191–212. Williams, A.M., Morilak, D.A., 1997. α1B adrenoceptors in rat paraventricular nucleus overlap with, but do not mediate, the induction of c-Fos expression by osmotic or restraint stress. Neuroscience 76, 901–913. Woodruff, M.L., Baisden, R.H., Whittington, D.L., 1986. Effects of electrical stimulation of the pontine A5 cell group on blood pressure and heart rate in the rabbit. Brain Res. 379, 10–23. Yamaguchi, K., 2013. Tachycardic responses to stimulation of β-adrenoceptors in the brain parenchyma in conscious rats. Neurosci. Res. 76, 213–223. Yokotani, K., Murakami, Y., Okada, S., Hirata, M., 2001. Role of brain arachidonic acid cascade on central CRF1 receptor-mediated activation of sympatho-adrenomedullary outflow in rats. Eur. J. Pharmacol. 419, 183–189. Yorimitsu, M., Okada, S., Yamaguchi-Shima, N., Shimizu, T., Arai, J., Yokotani, K., 2008. Role of brain adrenoceptors in the corticotropin-releasing factor-induced central activation of sympatho-adrenomedullary outflow in rats. Life Sci. 82, 487–494. Zhang, Z.H., Felder, R.B., 2008. Hypothalamic CRF and norepinephrine mediate sympathetic and cardiovascular responses to acute intracarotid injection of TNF-α in the rat. J. Neuroendocrinol. 20, 978–987. Zhang, Z.H., Yu, Y., Wei, S.G., Nakamura, K., Felder, R.B., 2011. EP3 receptors mediate PGE2induced hypothalamic paraventricular nucleus excitation and sympathetic activation. Am. J. Physiol 301, H1559–H1569.
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Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu
Possible role of adrenoceptors in the hypothalamic paraventricular nucleus in corticotropin-releasing factor-induced sympatho-adrenomedullary outflow in rats Shoshiro Okada ⁎, Naoko Yamaguchi Department of Pharmacology, Graduate School of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
a r t i c l e
i n f o
Article history: Received 7 September 2016 Received in revised form 30 December 2016 Accepted 20 January 2017 Keywords: Adrenoceptor Corticotropin-releasing factor Noradrenaline release Paraventricular nucleus of the hypothalamus Plasma catecholamines
a b s t r a c t Aims: A functional interaction between the corticotropin-releasing factor (CRF) system and noradrenergic neurons in the brain has been suggested. In the present study, we investigated the interrelationship between the central CRF-induced elevation of plasma catecholamines and adrenoceptor activation in the paraventricular nucleus of the hypothalamus (PVN) using urethane-anesthetized rats. Main methods: In rats under urethane anesthesia, a femoral venous line was inserted for infusion of saline, and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for the application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed with high-performance liquid chromatography with electrochemical detection. Quantification of noradrenaline in rat PVN microdialysates was performed with high-performance liquid chromatography with electrochemical detection. Key findings: We showed that centrally administered CRF elevated noradrenaline release in the PVN. Furthermore, we demonstrated that microinjection of phenylephrine into the PVN induced elevation of plasma levels of adrenaline, but not of noradrenaline, whereas microinjection of isoproterenol into the PVN induced elevation of plasma levels of noradrenaline, but not of adrenaline. Bilateral blockade of adrenoceptors in the PVN revealed that phentolamine significantly suppressed the CRF-induced elevation of plasma adrenaline level, while propranolol significantly CRF-induced elevation of plasma noradrenaline level. Significance: Our results suggest that centrally administered CRF-induced elevation of plasma levels of adrenaline and noradrenaline can be mediated via activation of α-adrenoceptors and β-adrenoceptors, respectively, in the rat PVN. © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Corticotropin-releasing factor (CRF), a 41-amino acid peptide, is a key stress-related neuropeptide and was originally established as the primary physiological regulator of adrenocorticotropic hormone (ACTH) secretion (Rivier and Vale, 1985). However, it has been well documented that the peptide exhibits a broad distribution in extrahypothalamic brain regions, acting as a neurotransmitter/ neuromodulator, and forms circuits in the brain (Sawchenko and Swanson, 1983; Cummings and Seybold, 1988). Increasing evidence indicates that within the central nervous system, the peptide also acts as a regulator of the autonomic nervous system and of cardiovascular function (Brown and Fisher, 1985; Brown et al., 1985; Fisher, 1988; Yokotani et al., 2001; Okada et al., 2003). Specifically, Brown et al. (1982) ⁎ Corresponding author at: Department of Pharmacology, Graduate School of Medicine, Aichi Medical University, Japan. E-mail address: [email protected] (S. Okada).
reported that intracerebroventricularly administered CRF produces an elevation of the plasma concentrations of noradrenaline and adrenaline in conscious rats. They revealed that CRF acts within the brain to stimulate sympathetic outflow by experiments with hypophysectomy, adrenalectomy and ganglion blocker, indicating that the central effects of CRF is thought to be entirely independent of ACTH release. By using selective receptor antagonists, we previously reported that the centrally administered CRF-induced elevation of plasma noradrenaline was mediated by the activation of α1 and β adrenoceptors in the brain, and that of plasma adrenaline is mediated by the activation of α1 adrenoceptors in the brain (Yorimitsu et al., 2008). More recently, we reported that intracerebroventricularly administered isoproterenol, a β adrenoceptor agonist, elevated plasma noradrenaline, but not adrenaline (Ando et al., 2015). Collectively, these observations suggest that centrally administered CRF might elevate plasma catecholamines via activation of brain adrenoceptors. Noradrenaline is one of the major neurotransmitters involved in brain function, and it acts as a warning signal under stress conditions.
http://dx.doi.org/10.1016/j.autneu.2017.01.008 1566-0702/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Okada, N. YamaguchiAutonomic Neuroscience: Basic and Clinical 203 (2017) 74–80
It activates α or β adrenoceptors in the brain to elicit several kinds of biological responses, such as increased blood pressure and heart rate and secretion of ACTH (Moore and Bloom, 1979; Woodruff et al., 1986; McCall, 1988; Schreihofer and Guyenet, 2000). The paraventricular nucleus of the hypothalamus (PVN) is a complex integrative center important for neurohumoral regulation and the maintenance of cardiovascular and body fluid homeostasis (Kenney et al., 2003; Benarroch, 2005). The PVN receives dense noradrenergic projections from the brainstem with an additional smaller contribution arising from the locus coeruleus (Sawchenko and Swanson, 1983). An ultrastructural study demonstrated that noradrenergic axon terminals contact gastric pre-autonomic neurons in the PVN (Balcita-Pedicino and Rinaman, 2007). Furthermore, functional analyses suggest that adrenoceptors in the PVN play an important role in regulating sympathetic outflow (Scheurink et al., 1990; Williams and Morilak, 1997; Chen et al., 2006; Zhang and Felder, 2008). Collectively, these observations suggest that noradrenergic neurons projecting to the PVN can activate adrenoceptors located in the PVN and can mediate the excitatory effects of noradrenaline to induce sympathetic outflow. In the present study, to identify the brain sites for CRF-induced sympatho-adrenomedullary outflow, we used a brain microdialysis technique to examine pharmacologically the possibility that centrally administered CRF can release noradrenaline in the PVN. Furthermore, the effects of microinjection into the PVN of phenylephrine, an α1adrenoceptor agonist, or isoproterenol, a β adrenoceptor agonist on the plasma levels of noradrenaline and adrenaline were examined. 2. Materials and methods 2.1. Animals Male Wistar rats weighing approximately 350 g were maintained in an air-conditioned room at 22–24 °C under a constant day-night rhythm for N2 weeks and given food (laboratory chow, CE-2; CLEA Japan, Hamamatsu, Japan) and water ad libitum. All experiments were conducted in compliance with the guiding principles for the care and use of laboratory animals approved by Aichi Medical University. 2.2. Microdialysis Microdialysis experiments were performed according to methods published in a previous report (Okada et al., 2000). Briefly, a stainless steel guide cannula held on the tip of an L-shaped stainless steel apparatus was implanted stereotaxically just above the right PVN. For intracerebroventricular administration of CRF, another stainless steel cannula (o.d. 0.35 mm) was inserted into the left lateral cerebral ventricle, and the peptide was applied in a volume of 10 μl using a 50-μl Hamilton syringe at the following coordinates (in mm): AP +1.59, L 1.5, V − 4.62 (AP, anterior from bregma; L, lateral from the midline; V, below the surface of the brain). The PVN was perfused with Ringer's solution (147 mM NaCl, 4 mM KCl and 2.3 mM CaCl2) at a flow rate of 2 μl/ min using a microinfusion pump (EP-60, Eicom, Kyoto, Japan). Three consecutive dialysates were used to measure the baseline release of noradrenaline. Noradrenaline was measured directly with high-performance liquid chromatography (HPLC) with electrochemical detection (Okada et al., 2002).
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− 1.8 mm; L, 0.3 mm; V, 7.6 mm: lateral ventricle: AP, − 0.8 mm; L, 1.5 mm; V, 4.0 mm, according to the rat brain atlas of Paxinos and Watson (2005). Four hours were allowed to elapse before the application of test substances. In the experimental group, a stainless steel injection cannula (o.d. 0.3 mm) was attached to PE-20 tubing, which was then connected to a Hamilton microsyringe (0.5-μl syringe for PVN injection; 10-μl syringe for intracerebroventricular injection). To examine effects of adrenoceptor agonists on plasma catecholamine levels, phenylephrine (5 or 10 nmol), isoproterenol (5 or 10 nmol) or sterile saline as a vehicle was injected unilaterally into the PVN in a volume of 50 nl over 30 s. Next, to examine effects of bilateral blockade of adrenoceptors in the PVN on CRF-induced elevation of plasma catecholamine levels, adrenoceptor antagonists in combination with CRF were administered. Phentolamine (6 nmol/side), propranolol (6 nmol/side) or dimethyl sulfoxide (DMSO) as a vehicle was injected bilaterally into the PVN in a volume of 100 nl over 60 s. After 30 min, CRF (1.5 nmol) or sterile saline was injected intracerebroventricularly in a volume of 10 μl over 30 s. At the end of the experiments, brains were removed, post-fixed in 4% paraformaldehyde solution and cut on a cryostat, and then brain sections were stained in hematoxylin solution for histological verification of injection sites. 2.4. Measurement of plasma catecholamines Arterial blood samples (250 μl) were collected in a heparinized tube through an arterial catheter and were preserved on ice during the experiments. Immediately after the final sampling, plasma was prepared by centrifugation (3000 ×g for 10 min at 4 °C). According our previous reports, catecholamines in the plasma were extracted by alumina and were assayed with HPLC with electrochemical detection (Ando et al., 2015). 2.5. Treatment of data and statistics Results are expressed as the mean ± S.E.M. of the net change above the respective basal value. The data were analyzed by Student's t-test (Fig. 1) or by one-way ANOVA with SPSS v22.0, followed by a posthoc analysis with the Bonferroni method (Figs. 2, 3, 5). P values b 0.05 were taken to indicate statistical significance.
2.3. Microinjection With rats under urethane anesthesia (1.2 g/kg, intraperitoneally), a femoral venous line was inserted for infusion of saline (1.2 ml/h), and an arterial line was inserted for collection of blood samples, as described previously (Ando et al., 2015). Next, the animal was placed in a stereotaxic apparatus, as described previously (Ando et al., 2015). Holes were drilled in the skull for administration of test substances. The stereotaxic coordinates of the tip of the cannula were as follows (in mm): PVN: AP,
Fig. 1. Effects of intracerebroventricularly (i.c.v.) administered CRF on the release of noradrenaline in the PVN. ●, CRF (n = 6); ○, vehicle (10 μl saline/animal, i.c.v.) (n = 4). *Significantly different (pb0.05) from vehicle.
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Fig. 2. Effects of PVN microinjection of the α1-adrenergic agonist phenylephrine on plasma levels of noradrenaline and adrenaline. Arrows indicate each PVN microinjection. Phenylephrine (5 and 10 nmol/animal) or vehicle (saline) was administered in a volume of 50 nl. ○, vehicle (saline) (n = 6); ●, phenylephrine (5 nmol/animal) (n = 6); ▲, phenylephrine (10 nmol/animal) (n = 6). *Significantly different (p b 0.05) from vehicle-treated control. Each point represents the mean ± S.E.M.
2.6. Drugs The following pharmacological agents were used: synthetic corticotropin-releasing hormone (rat/human) (Peptide Institute, Osaka, Japan); phenylephrine, isoproterenol and phentolamine (Sigma-Aldrich Chemicals, St. Louis, MO); propranolol (Wako Pure Chemical Industries, Osaka, Japan). All other reagents were the highest grade available (Nacalai Tesque, Kyoto, Japan).
3. Results 3.1. Effect of CRF on noradrenaline release in the PVN The mean baseline level of noradrenaline in the microdialysate (three consecutive samples) was 0.9 ± 0.1 pg/20 min-fraction (n = 70). Application of Ringer's solution into the PVN with a microdialysis probe did not alter the baseline release of noradrenaline in the PVN (Fig. 1). Application of CRF (1.5 nmol/animal) into the left ventricle elevated the release of noradrenaline in the PVN. After the application of CRF, the maximal response of noradrenaline release was observed at 80 min, and then it declined to the baseline level (Fig. 1).
3.2. Effects of phenylephrine, an α1-adrenoceptor agonist, on plasma catecholamine levels after microinjection into the PVN The 50 nl volume of saline microinjected into the PVN and blood sampling six times over a 60-min period had no significant effect on the baseline plasma levels of either noradrenaline or adrenaline (Fig. 2). Unilateral microinjection of phenylephrine (5 and 10 nmol/50 nl) directly into the PVN dose-dependently elevated the plasma levels of adrenaline, while it did not alter the plasma levels of noradrenaline (Fig. 2). The adrenaline levels peaked at 10 min and then gradually declined toward baseline. The actual values for noradrenaline and adrenaline at 0 min were 303.8 ± 67.9 and 394.4 ± 85.3 pg/ml, respectively, for the saline-treated group (n = 6), 493.6 ± 125.5 and 339.7 ± 44.2 pg/ml, respectively, for the 5 nmol phenylephrine-treated group (n = 6), and 338.7 ± 80.0 and 581.5 ± 133.4 pg/ml, respectively, for the 10 nmol phenylephrine-treated group (n = 6).
3.3. Effects of isoproterenol, a β-adrenoceptor agonist, on plasma catecholamine levels after microinjection into the PVN The 50 nl volume of saline microinjected into the PVN and blood sampling six times over a 60-min period had no significant effect on
Fig. 3. Effects of PVN microinjection of the β-adrenergic agonist isoproterenol on plasma levels of noradrenaline and adrenaline. Arrows indicate each PVN microinjection. Isoproterenol (5 and 10 nmol/animal) or vehicle (saline) was administered in a volume of 50 nl. ○, vehicle (saline) (n = 5); ●, isoproterenol (5 nmol/animal) (n = 7); ▲, isoproterenol (10 nmol/animal) (n = 8). *Significantly different (p b 0.05) from vehicle-treated control. Each point represents the mean ± S.E.M.
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the baseline plasma levels of either noradrenaline or adrenaline (Fig. 3). Unilateral microinjection of isoproterenol (10 nmol/50 nl) directly into the PVN significantly elevated the plasma levels of noradrenaline (Fig. 3). The noradrenaline levels peaked at 5 min and then gradually declined toward baseline. In contrast, only the highest dose of isoproterenol (10 nmol/animal) significantly elevated the plasma levels of adrenaline (Fig. 3). The actual values for noradrenaline and adrenaline at 0 min were 384.4 ± 38.9 and 263.8 ± 81.1 pg/ml, respectively, for the saline-treated group (n = 5), 271.9 ± 51.6 and 215.4 ± 72.8 pg/ml, respectively, for the 5 nmol isoproterenol-treated group (n = 7), and 351.9 ± 21.1 and 117.1 ± 31.4 pg/ml, respectively, for the 10 nmol isoproterenol-treated group (n = 8). 3.4. Histological verification of unilateral microinjection sites in the PVN A typical unilateral microinjection site in the PVN is shown in Fig. 4A. Diagrams showing microinjection sites in the PVN at different
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rostrocaudal levels (1.56 to 1.92 mm caudal to bregma) are depicted in Fig. 4B. In these diagrams, each symbol represents a microinjection site in one animal. Data from rats for which microinjection sites were confirmed were used for analysis. 3.5. Effects of bilateral microinjections of phentolamine, an α-adrenoceptor antagonist, or propranolol, a β-adrenoceptor antagonist, into the PVN on the CRF-induced elevation of plasma catecholamine levels The bilateral microinjections of vehicle-1 (100 nl of DMSO) into the PVN in combination with vehicle-2 (10 μl of saline) injected into the lateral ventricle had no significant effect on the plasma levels of catecholamines (Fig. 5). The bilateral microinjections of vehicle-1 (100 nl of DMSO) into the PVN in combination with CRF (1.5 nmol/10 μl) injected into the lateral ventricle had significantly elevated plasma catecholamine levels (Fig. 5). The bilateral microinjections of phentolamine (6 nmol/100 nl/side) into the PVN did not affect the CRF-induced
Fig. 4. Microinjection sites in the PVN. (A) Photomicrograph of a coronal rat brain section indicating the injection sites in the PVN. Scale bar = 200 μm. (B) Diagrammatic representation of rat brain frontal sections, from Paxinos and Watson (2005), showing the distribution of injection sites in phenylephrine (black) and isoproterenol (white) in the unilateral PVN of urethane-anesthetized rats (circle, vehicle; triangle, 5 nmol; square, 10 nmol). 3V, third ventricle; AH, anterior hypothalamic area; f, fornix; PVN, paraventricular nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.
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Fig. 5. Effects of bilateral microinjections of phentolamine, an α-adrenoceptor antagonist, or propranolol, a β-adrenoceptor antagonist into the PVN on the CRF-induced elevation of plasma catecholamine levels. Arrows indicate each PVN microinjection and intracerebroventricular administration. Phentolamine (6 nmol/side), propranolol (6 nmol/side) or vehicle-1 (DMSO) was microinjected bilaterally into the PVN in a volume of 100 nl. CRF (1.5 nmol/animal) or vehicle-2 (saline) was administered intracerebroventricularly in a volume of 10 μl. ○, vehicle1 + CRF (n = 6); ■, phentolamine + CRF (n = 5); ▲, propranolol + CRF (n = 5); ●, vehicle-1 + vehicle-2 (n-5). *Significantly different (p b 0.05) from vehicle-1 + CRF-treated group. Each point represents the mean ± S.E.M.
elevation of plasma noradrenaline level, but suppressed significantly the CRF-induced elevation of plasma adrenaline level (Fig. 5). In contrast, microinjections of propranolol (6 nmol/100 nl/side) into the PVN significantly suppressed the CRF-induced elevation of plasma noradrenaline level, while those of propranolol showed no statistically significant change on the CRF-induced elevation of plasma adrenaline level, although those of propranolol tended to suppress the elevation of plasma adrenaline level at 60 and 90 min (p = 0.092 at 60 min; p = 0.056 at 90 min). The actual values for noradrenaline and adrenaline at 0 min were 204.8 ± 65.6 and 151.9 ± 57.5 pg/ml for vehicle-1 plus vehicle-2-treated group (n = 5), 343.8 ± 40.4 and 210.8 ± 30.5 pg/ml for vehicle-1 plus CRF-treated group (n = 6), 346.8 ± 104.2 and 400.3 ± 124.1 pg/ml for phentolamine plus CRF-treated group (n = 5), and 289.4 ± 41.5 and 127.5 ± 55.2 pg/ml for propranolol plus CRF-treated group (n = 5), respectively. 3.6. Histological verification of bilateral microinjection sites in the PVN A typical bilateral microinjection site in the PVN is shown in Fig. 6A. Diagrams showing microinjection sites in the PVN at different rostrocaudal levels (1.56 to 1.92 mm caudal to bregma) are depicted in Fig. 6B. In these diagrams, each symbol represents a microinjection site in one animal. Data from rats for which microinjection sites were confirmed were used for analysis. 4. Discussion It has been shown that central administration of CRF induces autonomic activation, resulting in increases in blood pressure, heart rate and plasma catecholamines (Brown et al., 1982; Korte et al., 1993; Yokotani et al., 2001; Okada et al., 2003). Immunohistochemical analyses revealed that intracerebroventricularly administered CRF evokes Fos expression in the PVN, a key center for central autonomic control (Bittencourt and Sawchenko, 2000). Furthermore, several lines of evidence indicate that centrally administered CRF activates noradrenergic neurons projecting to the PVN and induces an increase in noradrenaline release in the PVN (Otagiri et al., 2000; Dunn et al., 2004; Okada et al., 2008). Collectively, these observations may imply a functional interaction between central CRF and noradrenergic neurons projecting to the PVN. However, the precise mechanisms underlying these actions of CRF are still not fully defined.
In the present study, we showed that centrally administered CRF increased noradrenaline release in the PVN in urethane-anesthetized rats, consistent with previous reports using different experimental conditions (Dunn et al., 2004; Okada et al., 2008). Thus, it is conceivable that increased noradrenaline released in the PVN might stimulate adrenoceptors located in the PVN. Since there is considerable evidence that adrenoceptors are abundant in the PVN (Scriabine et al., 1976; McCall and Humphrey, 1981), we next performed microinjection experiments with α and β adrenoceptor agonists. Interestingly, α-adrenoceptor activation by phenylephrine increased plasma levels of adrenaline, whereas βadrenoceptor activation by isoproterenol increased plasma levels of noradrenaline. Therefore, their physiological roles must be different. It has been reported that bilateral microinjection of phenylephrine directly into the PVN increases renal sympathetic activity and heart rate, but it did not affect mean arterial pressure (Zhang and Felder, 2008). More recently, Yamaguchi (2013) demonstrated that administration of isoproterenol into the PVN increased the heart rate in freely moving rats. Collectively, these lines of evidence indicate that stimulation of α or β-adrenoceptor located in the PVN can induce sympathetic outflow. The present study clearly demonstrated that activation of α1adrenoceptors and β-adrenoceptors in the PVN could separately regulate plasma levels of catecholamines in urethane-anesthetized rats. Finally, to reveal whether adrenoceptors located in the PVN mediate the central action of CRF to increase plasma catecholamine, we next examined the effects of α or β adrenoceptor antagonists microinjected into the PVN on the CRF-induced elevations of plasma levels of catecholamine. The present results clearly demonstrated that central CRF-induced elevation of noradrenaline was mediated via activation of β adrenoceptor in the PVN, while that of adrenaline was mediated via activation of α adrenoceptor in the PVN. Thus, it would be reasonable to assume that the endogenous noradrenaline released by CRF stimulation binds to adrenoceptors in the PVN, and that α or β adrenoceptor in the PVN could play differential roles for CRF-induced elevations of plasma catecholamines. Although Itoi et al. (1994) showed that microinjection of noradrenaline into the PVN stimulates CRF secretion into the portal circulation to promote hypothalamic-adrenal axis via activation of α1-adrenergic receptors but not of β adrenergic receptors, there has been no detailed studies regarding roles for adrenoceptors in the PVN responsible for central action of CRF-induced elevation of plasma catecholamine. In light of the present study, one can assume that activation of noradrenergic neurons projecting to the PVN might promote not only
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Fig. 6. Bilateral microinjection sites in the PVN. (A) Photomicrograph of a coronal rat brain section indicating the injection sites in the PVN. Scale bar = 200 μm. (B) Diagrammatic representation of rat brain frontal sections, from Paxinos and Watson (2005), showing the distribution of injection sites. ○, vehicle-1 + CRF; ■, phentolamine + CRF; ▲, propranolol + CRF; ●, vehicle-1 + vehicle-2. 3V, third ventricle; AH, anterior hypothalamic area; f, fornix; PVN, paraventricular nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus.
hypothalamic-adrenal axis but also sympatho-adrenomedullary outflow via α or β adrenoceptors. Taking physiological roles for CRF as a stress related peptide into account, the present results may give an opportunity to reveal the detailed mechanisms underlying CRF-induced stress responses. Previously, we reported that central CRF differentially elevated plasma levels of catecholamines, namely, noradrenaline via brain prostaglandin E2 (PGE2) and adrenaline via brain thromboxane A2 (Okada et al., 2003). Recently, a microinjection study revealed that EP3 receptors mediate the central excitatory effects of PGE2 on PVN neurons and sympathetic discharge, such as renal nerve activity, mean blood pressure and heart rate (Zhang et al., 2011). More recently, we reported that centrally administered isoproterenol increased plasma levels of noradrenaline, but not of adrenaline, via PGE2-mediated mechanisms (Ando et al., 2015). These observations raised the possible connections between adrenoceptor activation and mode of action of prostanoids in the PVN. Further analysis of the detailed mechanisms underlying these interactions will be carried out in future studies.
Our previous study demonstrated that CRF-induced sympathoadrenomedullary outflow is mediated by central CRF1 receptors (Yokotani et al., 2001). Although it has been shown that one of the first anatomical sites exposed to centrally-administered CRF is the lateral septum mediolateral nucleus (Liu et al., 2004; Gallagher et al., 2008), the precise brain sites responsible for the various actions of CRF are still unknown. Interestingly, it has been demonstrated that expression of CRF1 receptor mRNA in the PVN is localized principally to autonomic-related aspects of the parvocellular division (Bittencourt and Sawchenko, 2000; Van Pett et al., 2000). In addition, recent reports suggest a possible involvement of presynaptic CRF1 receptors in modulating neurotransmitter release (Gallagher et al., 2008; Kirby et al., 2008; Nie et al., 2009). Collectively, these observations raise an interesting possibility that CRF1 receptors may exist and act on noradrenergic terminals projecting to the PVN to regulate noradrenaline release. In conclusion, we showed that centrally administered CRF elevated noradrenaline release in the PVN. The increased noradrenaline in the PVN might activate α1-adrenoceptors and β-adrenoceptors located in
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