Stimulation of the hypothalamic arcuate nucleus increases brown adipose tissue nerve activity via hypothalamic paraventricular and dorsomedial nuclei.

Hypothalamic arcuate nucleus (ARCN) stimulation elicited increases in sympathetic nerve activity (IBATSNA) and temperature (TBAT) of interscapular brown adipose tissue (IBAT). The role of hypothalamic dorsomedial (DMN) and paraventricular (PVN) nuclei in mediating these responses was studied in urethane-anesthetized, artificially ventilated, male Wistar rats. In different groups of rats, inhibition of neurons in the DMN and PVN by microinjections of muscimol attenuated the increases in IBATSNA and TBAT elicited by microinjections of N-methyl-d-aspartic acid into the ipsilateral ARCN. In other groups of rats, blockade of ionotropic glutamate receptors by combined microinjections of D(-)-2-amino-7-phosphono-heptanoic acid (D-AP7) and NBQX into the DMN and PVN attenuated increases in IBATSNA and TBAT elicited by ARCN stimulation. Blockade of melanocortin 3/4 receptors in the DMN and PVN in other groups of rats resulted in attenuation of increases in IBATSNA and TBAT elicited by ipsilateral ARCN stimulation. Microinjections of Fluoro-Gold into the DMN resulted in retrograde labeling of cells in the ipsilateral ARCN, and some of these cells contained proopiomelanocortin (POMC), α-melanocyte-stimulating hormone (α-MSH), or vesicular glutamate transporter-3. Since similar projections from ARCN to the PVN have been reported by us and others, these results indicate that neurons containing POMC, α-MSH, and glutamate project from the ARCN to the DMN and PVN. Stimulation of ARCN results in the release of α-MSH and glutamate in the DMN and PVN which, in turn, cause increases in IBATSNA and TBAT.

Attenuation of angiotensin type 2 receptor function in the rostral ventrolateral medullary pressor area of the spontaneously hypertensive rat.

We hypothesized that blockade of angiotensin II type 2 receptors (AT2Rs) in the rostral ventrolateral medullary pressor area (RVLM) may elicit sympathoexcitatory responses which are smaller in hypertensive rats compared to normotensive rats. This hypothesis was tested in urethane-anesthetized, artificially ventilated male 14-week-old spontaneously hypertensive rats (SHR). Age-matched male Wistar-Kyoto rats (WKY) and Wistar rats were used as controls. PD123319 (AT2R antagonist) was microinjected into the RVLM and mean arterial pressure (MAP), heart rate (HR) and greater splanchnic nerve activity (GSNA) were recorded. Increases in MAP, HR and GSNA elicited by unilateral microinjections of PD123319 into the RVLM were significantly smaller in SHR when compared with those in WKY and Wistar rats. Unilateral microinjections of l-glutamate (l-Glu) into the RVLM elicited greater increases in MAP and GSNA in SHR compared to those in WKY. AT2R immunoreactivity was demonstrated in the RVLM neurons which were retrogradely labeled from the intermediolateral cell column (IML) of the spinal cord. These results indicate that AT2Rs are present on the RVLM neurons projecting to the IML and their blockade results in sympathoexcitatory responses. Activation of AT2Rs has an inhibitory influence in the RVLM and these receptors are tonically active. Attenuation of the function of AT2Rs in the RVLM may play a role in genesis and/or maintenance of hypertension in SHR.

GABA and glycine receptors in the nucleus ambiguus mediate tachycardia elicited by chemical stimulation of the hypothalamic arcuate nucleus.

We have previously reported that stimulation of the hypothalamic arcuate nucleus (ARCN) by microinjections of N-methyl-d-aspartic acid (NMDA) elicits tachycardia, which is partially mediated via inhibition of vagal inputs to the heart. The neuronal pools and neurotransmitters in them mediating tachycardia elicited from the ARCN have not been identified. We tested the hypothesis that the tachycardia elicited from the ARCN may be mediated by inhibitory neurotransmitters in the nucleus ambiguus (nAmb). Experiments were done in urethane-anesthetized, artificially ventilated, male Wistar rats. In separate groups of rats, unilateral and bilateral microinjections of muscimol (1 mM), gabazine (0.01 mM), and strychnine (0.5 mM) into the nAmb significantly attenuated tachycardia elicited by unilateral microinjections of NMDA (10 mM) into the ARCN. Histological examination of the brains showed that the microinjections sites were within the targeted nuclei. Retrograde anatomic tracing from the nAmb revealed direct bilateral projections from the ARCN and hypothalamic paraventricular nucleus to the nAmb. The results of the present study suggest that tachycardia elicited by stimulation of the ARCN by microinjections of NMDA is mediated via GABAA and glycine receptors located in the nAmb.

Cardiovascular responses to microinjections of endomorphin-2 into the nucleus of the solitary tract are attenuated in the spontaneously hypertensive rat.

Stimulation of µ1-opioid receptors (M1ORs) in the medial nucleus solitarius (mNTS) by endomorphin-2 (EM2) elicits decreases in mean arterial pressure (MAP), heart rate (HR) and greater splanchnic nerve activity (GSNA) in Wistar rats. We tested the hypothesis that EM2-induced responses in the mNTS may be attenuated in the spontaneously hypertensive rat (SHR). Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male SHR and Wistar-Kyoto rats (WKY). Alterations in responses to chemical stimulation of the hypothalamic arcuate nucleus (ARCN) after bilateral blockade of M1ORs in the mNTS were also studied. In SHR, microinjections of EM2 into the mNTS elicited smaller decreases in MAP, HR and GSNA compared to those elicited in WKY; smaller cardiovascular responses in SHR can be explained by lower expression of M1OR mRNA in the NTS of SHR compared to WKY. Decreases in MAP and GSNA and increases in HR were elicited by microinjections of N-methyl-D-aspartic acid (NMDA) into the ARCN of WKY. Bilateral blockade of M1ORs in the mNTS attenuated the decreases in MAP and GSNA and exaggerated the increases in HR elicited by the ARCN stimulation in WKY but not in SHR. Tonic inhibitory activity of neuropeptide Y/gamma-aminobutyric acid (NPY/GABA) neurons in the ARCN is attenuated in SHR; this observation may explain increases in MAP, GSNA and HR elicited by microinjections of NMDA into the ARCN of SHR. These results demonstrate that attenuation of EM2-induced responses in the mNTS of SHR may contribute to the excitatory responses elicited by ARCN stimulation in SHR.

Cardiovascular effect of angiotensin-(1-12) in the caudal ventrolateral medullary depressor area of the rat.

Angiotensin (ANG)-(1-12) excites neurons via ANG II type 1 receptors (AT1Rs), which are present in the caudal ventrolateral medullary depressor area (CVLM). We hypothesized that microinjections of ANG-(1-12) into the CVLM may elicit decreases in mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity. This hypothesis was tested in urethane-anesthetized adult male Wistar rats. Microinjections of ANG-(1-12) into the CVLM elicited decreases in MAP, HR, and greater splanchnic nerve activity (GSNA). ANG-(1-12)-induced responses consisted of initial (first 1-8 min) and delayed (8-24 min) phases. Prior microinjections of losartan, A-779, and captopril into the CVLM blocked initial, delayed, and both phases of ANG-(1-12) responses, respectively. Blockade of GABA receptors in the rostral ventrolateral medullary pressor area (RVLM) attenuated cardiovascular responses elicited by microinjections of ANG-(1-12) into the ipsilateral CVLM. Microinjections of ANG-(1-12) into the CVLM potentiated the reflex decreases and attenuated the reflex increases in GSNA elicited by intravenous injections of phenylephrine and sodium nitroprusside, respectively. These results indicate that microinjections of ANG-(1-12) into the CVLM elicit decreases in MAP, HR, and GSNA. Initial and delayed phases of these responses are mediated via ANG II and ANG-(1-7), respectively; the effects of ANG II and ANG-(1-7) are mediated via AT1Rs and Mas receptors, respectively. Captopril blocked both phases of ANG-(1-12) responses, indicating that angiotensin-converting enzyme is important in mediating these responses. GABA receptors in the RVLM partly mediate the cardiovascular responses to microinjections of ANG-(1-12) into the CVLM. Microinjections of ANG-(1-12) into the CVLM modulate baroreflex responses.

Activation of melanocortin receptors in the intermediolateral cell column of the upper thoracic cord elicits tachycardia in the rat.

Melanocortin receptors (MCRs) are present in the intermediolateral cell column of the spinal cord (IML). We tested the hypothesis that activation of MCRs in the IML elicits cardioacceleratory responses and the source of melanocortins in the IML may be the melanocortin-containing neurons in the hypothalamic arcuate nucleus (ARCN). Experiments were done in urethane-anesthetized, artificially ventilated adult male Wistar rats. Microinjections (50 nl) of α-melanocyte stimulating hormone (α-MSH) (0.4-2 mM) and adrenocorticotropic hormone (ACTH) (0.5-2 mM) into the right IML elicited increases in heart rate (HR). These tachycardic responses were blocked by microinjections of melanocortin receptor 4 (MC4R) antagonists [SHU9119 (0.25 mM) or agouti-related protein (AGRP, 0.1 mM)] into the right IML. Stimulation of right ARCN by microinjections (30 nl) of N-methyl-d-aspartic acid (NMDA, 10 mM) elicited increases in HR. Blockade of MC4Rs in the ipsilateral IML at T1-T3 using SHU9119 (0.25 mM) attenuated the tachycardic responses elicited by subsequent microinjections of NMDA into the ipsilateral ARCN. ARCN neurons retrogradely labeled by microinjections of Fluoro-Gold into the right IML showed immunoreactivity for proopiomelanocortin (POMC), α-MSH, and ACTH. Fibers immunoreactive for POMC, α-MSH, and ACTH were present in the IML at T1-T3. These results indicated that activation of MC4Rs in the right IML elicited tachycardia and one of the sources of melanocortins in the IML is the ARCN. Melanocortin levels are elevated in stress and ARCN neurons are activated during stress. Our results allude to the possibility that cardiac effects of stress may be mediated via melanocortin containing ARCN neurons that project to the IML.

Mechanisms of cardiovascular actions of urocortins in the hypothalamic arcuate nucleus of the rat.

The presence of urocortins (UCNs) and corticotropin-releasing factor (CRF) receptors has been reported in the hypothalamic arcuate nucleus (ARCN). We have previously reported that UCNs are involved in central cardiovascular regulation. Based on this information, we hypothesized that the ARCN may be one of the sites where UCNs exert their central cardiovascular actions. Experiments were done in artificially ventilated, adult male Wistar rats anesthetized with urethane. Unilateral microinjections (30 nl) of UCN1 (0.12-2 mM) elicited decreases in mean arterial pressure (MAP) and heart rate (HR). Maximum cardiovascular responses were elicited by a 1 mM concentration of UCN1. Microinjections of UCN2 and UCN3 (1 mM each) into the ARCN elicited similar decreases in MAP and HR. UCN1 was used as a prototype for the other experiments described below. HR responses elicited by UCN1 were significantly attenuated by bilateral vagotomy. Prior microinjections of NBI-27914 (CRF-1 receptor antagonist) and astressin (CRF-1 receptor and CRF-2 receptor antagonist) (1 mM each) into the ARCN significantly attenuated the cardiovascular responses elicited by UCN1 microinjections at the same site. Microinjections of UCN1 into the ARCN decreased efferent renal sympathetic nerve activity. It was concluded that microinjections of UCN1, UCN2, and UCN3 into the ARCN elicited decreases in MAP and HR. Decreases in MAP, HR, and renal sympathetic nerve activity elicited by UCN1 microinjections into the ARCN were mediated via CRF receptors. Bradycardic responses to UCN1 were mediated via the activation of vagus nerves, and decreases in MAP may be mediated via decreases in sympathetic nerve activity.

Angiotensin-(1-12) in the rostral ventrolateral medullary pressor area of the rat elicits sympathoexcitatory responses.

The rostral ventrolateral medullary pressor area (RVLM) is known to be critical in the regulation of cardiovascular function. In this study, it was hypothesized that the RVLM may be one of the sites of cardiovascular actions of a newly discovered angiotensin, angiotensin-(1-12) [Ang-(1-12)]. Experiments were carried out in urethane-anaesthetized, artificially ventilated, adult male Wistar rats. The RVLM was identified by microinjections of L-glutamate (5 mM). The volume of all microinjections into the RVLM was 100 nl. Microinjections of Ang-(1-12) (0.1-1.0 mM) into the RVLM elicited increases in mean arterial pressure and heart rate. Maximal cardiovascular responses were elicited by 0.5 mM Ang-(1-12); this concentration was used in the other experiments described. Microinjections of Ang-(1-12) increased greater splanchnic nerve activity. The tachycardic responses to Ang-(1-12) were not altered by bilateral vagotomy. The cardiovascular responses elicited by Ang-(1-12) were attenuated by microinjections of an angiotensin II type 1 receptor (AT(1)R) antagonist (losartan), but not an AT(2)R antagonist (PD123319), into the RVLM. Combined inhibition of angiotensin-converting enzyme and chymase in the RVLM abolished Ang-(1-12)-induced responses. Angiotensin-(1-12)-immunoreactive cells were present in the RVLM. Angiotensin II type 1 receptors and phenylethanolamine-N-methyl-transferase were present in the RVLM neurons retrogradely labelled by microinjections of Fluoro-Gold into the intermediolateral cell column of the thoracic spinal cord. Angiotensin-(1-12)-containing neurons in the hypothalamic paraventricular nucleus did not project to the RVLM. These results indicated that: (1) microinjections of Ang-(1-12) into the RVLM elicited increases in mean arterial pressure, heart rate and greater splanchnic nerve activity; (2) both angiotensin-converting enzyme and chymase were needed to convert Ang-(1-12) into angiotensin II; and (3) AT(1)Rs, but not AT(2)Rs, in the RVLM mediated the Ang-(1-12)-induced responses.

Bradycardic effects of microinjections of urocortin 3 into the nucleus ambiguus of the rat.

The presence of urocortin 3 (UCN3) and CRF2 receptors (CRF2R) has been demonstrated in brain tissue. Nucleus ambiguus (nAmb) is the predominant brain area providing parasympathetic innervation to the heart. On the basis of these reports, it was hypothesized that activation of CRF2Rs in the nAmb may elicit cardiac effects. Experiments were carried out in urethane-anesthetized, artificially ventilated, and adult male Wistar rats. Microinjections of l-glutamate (l-GLU, 5 mM) were used to identify the nAmb. Different concentrations of UCN3 (0.031, 0.062, 0.125, 0.25, and 0.5 mM) microinjected into the nAmb elicited decreases in heart rate (HR) (5.3 ± 1, 22 ± 3.3, 38 ± 4.9, 45.7 ± 2.7, and 27.3 ± 2.3 bpm, respectively). The volume of all microinjections was 30 nl. Blood pressure changes concomitant with decreases in HR were not observed. Bradycardia elicited by microinjections of UCN3 (0.25 mM; maximally effective concentration) into the nAmb was significantly (P < 0.05) attenuated by microinjections of selective CRF2R antagonists (K41498, 0.5 mM, and astressin 2B, 0.25 mM) at the same site. Bilateral vagotomy abolished the bradycardic responses to UCN3. These results indicated that activation of CRF2Rs in the nAmb by UCN3 elicited bradycardia, which was vagally mediated. UCNs have been reported to exert cardioprotective effects in heart failure and ischemia/reperfusion injury. In this situation, centrally induced bradycardia by UCN3 would be beneficial. The results of the present investigation provide a platform for future studies on the role of CRF2Rs in the nAmb in pathological states such as heart failure.

Cardiovascular actions of angiotensin-(1-12) in the hypothalamic paraventricular nucleus of the rat are mediated via angiotensin II.

The role of the hypothalamic paraventricular nucleus (PVN) in cardiovascular regulation is well established. In this study, it was hypothesized that the PVN may be one of the sites of cardiovascular actions of a newly discovered angiotensin, angiotensin-(1-12). Experiments were carried out in urethane-anaesthetized, artificially ventilated, adult male Wistar rats. The PVN was identified by microinjections of NMDA (10 mm). Microinjections (50 nl) of angiotensin-(1-12) (1 mm) into the PVN elicited increases in mean arterial pressure, heart rate and renal sympathetic nerve activity. The tachycardic responses to angiotensin-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by angiotensin-(1-12) were attenuated by microinjections of an angiotensin II type 1 receptor (AT(1)R) antagonist (losartan), but not an angiotensin II type 1 receptor (AT(2)R) antagonist (PD123319), into the PVN. Combined inhibition of angiotensin-converting enzyme and chymase in the PVN abolished angiotensin-(1-12)-induced responses. Angiotensin-(1-12)-immunoreactive cells and fibres were more numerous in the middle and caudal regions of the PVN. Angiotensin-(1-12) was present in many, but not all, vasopressinergic PVN cells. This peptide was also present in some non-vasopressinergic PVN cells, but not in oxytocin-containing PVN cells. These results can be summarized as follows: (1) microinjections of angiotensin-(1-12) into the PVN elicited increases in mean arterial pressure, heart rate and renal sympathetic nerve activity; (2) heart rate responses were mediated via both sympathetic and vagus nerves; (3) both angiotensin-converting enzyme and chymase were needed to convert angiotensin-(1-12) to angiotensin II in the PVN; and (4) AT(1)Rs, but not AT(2)Rs, in the PVN mediated angiotensin-(1-12)-induced responses. It was concluded that the cardiovascular actions of angiotensin-(1-12) in the PVN are mediated via its conversion to angiotensin II.

Cardiovascular responses elicited by a new endogenous angiotensin in the nucleus tractus solitarius of the rat.

Cardiovascular effects of angiotensin-(1-12) [ANG-(1-12)] were studied in the medial nucleus of the tractus solitarius (mNTS) in anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of ANG-(1-12) (0.06 mM) into the mNTS elicited maximum decreases in mean arterial pressure (MAP; 34 ± 5.8 mmHg) and heart rate (HR; 39 ± 3.7 beats/min). Bilateral vagotomy abolished ANG-(1-12)-induced bradycardia. Efferent greater splanchnic nerve activity was decreased by microinjections of ANG-(1-12) into the mNTS. Blockade of ANG type 1 receptors (AT(1)Rs; using ZD-7155 or L-158,809), but not ANG type 2 receptors (AT(2)Rs; using PD-123319), significantly attenuated ANG-(1-12)-induced cardiovascular responses. Simultaneous inhibition of both angiotensin-converting enzyme (ACE; using captopril) and chymase (using chymostatin) completely blocked the effects of ANG-(1-12). Microinjections of A-779 [ANG-(1-7) antagonist] did not attenuate ANG-(1-12)-induced responses. Pressure ejection of ANG-(1-12) (0.06 mM, 2 nl) caused excitation of barosensitive mNTS neurons, which was blocked by prior application of the AT(1)R antagonist. ANG-(1-12)-induced excitation of mNTS neurons was also blocked by prior sequential applications of captopril and chymostatin. These results indicate that 1) microinjections of ANG-(1-12) into the mNTS elicited depressor and bradycardic responses by exciting barosensitive mNTS neurons; 2) the decreases in MAP and HR were mediated via sympathetic and vagus nerves, respectively; 3) AT(1)Rs, but not AT(2)Rs, mediated these actions of ANG-(1-12); 4) the responses were mediated via the conversion of ANG-(1-12) to ANG II and both ACE and chymase were involved in this conversion; and 5) ANG-(1-7) was not one of the metabolites of ANG-(1-12) in the mNTS.

Microinjections of urocortin1 into the nucleus ambiguus of the rat elicit bradycardia.

Urocortins are members of the hypothalamic corticotropin-releasing factor (CRF) peptide family. Urocortin1 (UCN1) mRNA has been reported to be expressed in the brainstem neurons. The present investigation was carried out to test the hypothesis that microinjections of UCN1 into the nucleus ambiguus (nAmb) may elicit cardiac effects. Urethane-anesthetized, artificially ventilated, adult male Wistar rats, weighing between 300-350 g, were used. nAmb was identified by microinjections of l-glutamate (5 mM, 30 nl). Microinjections (30 nl) of different concentrations (0.062, 0.125, 0.25, and 0.5 mM) of UCN1 into the nAmb elicited bradycardic responses (26.5 ± 1, 30.1 ± 1.7, 46.9 ± 1.7, and 40.3 ± 2.6 beats/min, respectively). These heart rate responses were not accompanied by significant changes in mean arterial pressure. The bradycardic responses to maximally effective concentration of UCN1 (0.25 mM) were significantly (P < 0.05) attenuated by prior microinjections of a selective antagonist (NBI 27914, 1.5 mM) for CRF type 1 receptor (CRF1R). Prior microinjections of ionotropic glutamate receptor (iGLUR) antagonists [d-(-)-2-amino-7-phosphono-heptanoic acid and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-(f)quinoxaline-7-sulfonamide disodium] also attenuated the bradycardia elicited by UCN1 microinjections into the nAmb. Microinjections of NBI 27914 (1.5 mM) into the nAmb did not alter baroreflex responses. Bilateral vagotomy abolished the bradycardic responses to microinjections of UCN1 into the nAmb. These results indicated that 1) microinjections of UCN1 into the nAmb elicited bradycardia, 2) the bradycardia was vagally mediated, 3) activation of CRF1Rs in the nAmb was responsible for the actions of UCN1, and 4) activation of iGLURs in the nAmb also participated in the bradycardia elicited by UCN1.

The hypothalamic arcuate nucleus: a new site of cardiovascular action of angiotensin-(1-12) and angiotensin II.

The hypothalamic arcuate nucleus (ARCN) has been reported to play a significant role in cardiovascular regulation. It has been hypothesized that the ARCN may be one of the sites of cardiovascular actions of angiotensins (ANGs). Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats. The ARCN was identified by microinjections of N-methyl-d-aspartic acid (NMDA; 10 mM). Microinjections (50 nl) of ANG-(1-12) (1 mM) into the ARCN elicited increases in mean arterial pressure (MAP), heart rate (HR), and greater splanchnic nerve activity (GSNA). The tachycardic responses to ANG-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by ANG-(1-12) were attenuated by microinjections of ANG II type 1 receptor (AT(1)R) antagonists but not ANG type 2 receptor (AT(2)R) antagonist. Combined inhibition of ANG-converting enzyme (ACE) and chymase in the ARCN abolished ANG-(1-12)-induced responses. Microinjections of ANG II (1 mM) into the ARCN also increased MAP and HR. Inhibition of ARCN by microinjections of muscimol (1 mM) attenuated the pressor and tachycardic responses to intravenously administered ANG-(1-12) and ANG II (300 pmol/kg each). These results indicated that 1) microinjections of ANG-(1-12) into the ARCN elicited increases in MAP, HR, and GSNA; 2) HR responses were mediated via both sympathetic and vagus nerves; 3) AT(1)Rs, but not AT(2)Rs, in the ARCN mediated ANG-(1-12)-induced responses; 4) both ACE and chymase were needed to convert ANG-(1-12) to ANG II in the ARCN; and 5) ARCN plays a role in mediating the cardiovascular responses to circulating ANGs.

Bradycardia elicited by microinjections of nociceptin/orphanin FQ into the intermediolateral cell column at T1-T2 in the rat.

Microinjections (30 nl) of nociceptin/orphanin FQ (N/OFQ) into the intermediolateral cell column (IML) at T1 and T2 levels of the spinal cord elicited bradycardia. The decreases in HR were 12.3+/-2.9, 17.3+/-2.7, 26.7+/-3.1, and 18.6+/-3.4 beats/min in response to 0.075, 0.15, 0.62, and 1.25 mM concentrations, respectively. Maximally effective concentration of N/OFQ was 0.62 mM. No changes in BP were elicited by microinjections of N/OFQ into the IML at T1-T2. The bradycardic responses were completely blocked by prior microinjections of a N/OFQ receptor (NOP receptor) antagonist ([N-phe(1)]-nociceptin-(1-13)-NH(2), 9 mM) into the IML at T1-T2. Blockade of myocardial beta-1 adrenergic receptors also abolished the bradycardic responses elicited by microinjections of N/OFQ into the IML. It was concluded that activation of NOP receptors in right IML at T1-T2 by N/OFQ elicited bradycardic responses which were mediated via the sympathetic nervous system.

Endomorphin-2 in the medial NTS attenuates the responses to baroreflex activation.

We have previously reported that microinjections of endomorphin-2 (E-2; an endogenous mu-receptor agonist) into the medial subnucleus of the NTS (mNTS) elicit depressor and bradycardic responses via activation of ionotropic glutamate receptors located on secondary mNTS-neurons. Based on this report, it was hypothesized that activation of secondary mNTS neurons by E-2 may result in an exaggeration of baroreflex responses. In order to test this hypothesis, baroreflex responses were studied in adult, urethane-anesthetized, artificially ventilated, male Wistar rats before and after the microinjections of E-2 into the mNTS. Baroreceptors were stimulated by applying pressure increments (80-100 mm Hg) in the carotid sinus and by electrical stimulation (stimulus intensity: 0.5 V, frequencies 5, 10, and 25 pulses/s, pulse duration: 1 ms) of the aortic nerve for 30-s periods. Baroreceptor stimulation elicited depressor and bradycardic responses. Microinjections (100 nl) of E-2 (0.4 mmol/l) into the mNTS attenuated the baroreflex responses. Microinjections of naloxone (an opioid receptor antagonist) into the mNTS (0.5 mmol/l) did not alter baroreflex responses. Based on these results, it was concluded that activation of mu-opioid receptors in the mNTS attenuates baroreflex responses. Possible mechanisms for excitatory effects of E-2 in the mNTS resulting in depressor and bradycardic responses, on one hand, and inhibitory effects resulting in attenuation of baroreflex responses, on the other, are discussed.

Cardiovascular effects of adrenocorticotropin microinjections into the rostral ventrolateral medullary pressor area of the rat.

The presence of adrenocorticotropic hormone (ACTH)-immunoreactive cells and melanocortin (MC) receptors (MC4 and to a lesser extent MC3) has been demonstrated in the medullary reticular formation in the general area where rostral ventrolateral medullary pressor area (RVLM) is located. The importance of RVLM in the regulation of cardiovascular function is well established. Based on these reports, it was hypothesized that ACTH may play a role in the regulation of cardiovascular function. To test this hypothesis, experiments were carried out on artificially ventilated, adult male, urethane-anesthetized and unanesthetized mid-collicular decerebrate rats. The RVLM was identified by microinjections (100 nl) of L-glutamate (L-Glu). Microinjections (100 nl) of ACTH (0.5, 1 and 2 mmol/l) into the RVLM elicited increases in MAP and HR; tachycardic responses were relatively inconsistent. The effects of ACTH were blocked by SHU9119 and agouti-related protein (AGRP). SHU9119 (a synthetic compound) and AGRP (an endogenous peptide) are antagonists for MC4, and to a lesser extent MC3, receptors. The specificity of these antagonists for MC receptors was indicated by their lack of effect on l-Glu responses. Microinjection of ACTH into the RVLM increased the efferent discharge in the greater splanchnic nerve. It was concluded that (1) ACTH exerts excitatory effects on RVLM neurons resulting in pressor and tachycardic responses, (2) these responses were mediated via MC4 and to a lesser extent MC3 receptors in the RVLM, and (3) the pressor effects of ACTH were mediated via sympathetic activation. This is the first report showing central cardiovascular actions of ACTH.

Microinjections of nociceptin into the nucleus ambiguus elicit tachycardia in the rat.

Cardiovascular effects of activation of opioid receptor like receptors (ORL1 receptors) in the nucleus ambiguus were studied in urethane-anesthetized, adult male Wistar rats. Microinjections of nociceptin (0.31, 0.62, 1.25 and 2.25 mmol/L) into the nucleus ambiguus elicited increases in heart rate (17.5 +/- 4, 33.3 +/- 2.9, 16.5 +/- 1.5 and 13.9 +/- 2.7 beats/min, respectively) which were blocked by an ORL1 receptor antagonist. These results indicate that activation of ORL1 receptors in the nucleus ambiguus elicits tachycardia.

Cardiovascular responses to activation of metabotropic glutamate receptors in the nTS of the rat.

Although several agonists and antagonists for different subtypes of metabotropic glutamate receptors (mGLURs) have become available in recent years, detailed information regarding their selectivity is not complete in the in vivo animal models. The purpose of the present investigation was to study the cardiovascular effects of microinjections of some of these mGLUR agonists and antagonists into the nucleus tractus solitarius (nTS). Microinjections (100 nl) of EC(50) concentrations of 3,5-DHPG (0.005 mM; mGLUR(1) agonist), APDC (17.3 mM; mGLUR(2/3) agonist), PPG (11.7 mM; mGLUR(8) agonist) and L-AP(4) (1 mM; mGLUR(4) agonist) into the nucleus tractus solitarius of urethane-anesthetized male Wistar rats elicited depressor and bradycardic responses which may be mediated by pre- and/or postsynaptic mechanisms. The blocking effect of mGLUR antagonists used here was not specific for any one type of glutamate receptors (GLURs). For example, AIDA (50 mM; mGLUR(1) antagonist) blocked the effects of EC(50) concentrations of 3,5-DHPG, and PPG. LY341495 (135 mM; mGLUR(2/3) antagonist) blocked all of the mGLURs and ionotropic GLURs. EGLU, APICA and MCCG (250 mM each; mGLUR(2/3) antagonists) blocked the effects of APDC, NMDA and AMPA. CPPG (80 mM) and MSOP (125 mM), mGLUR(4) antagonists, blocked the effects of 3,5-DHPG, PPG and L-AP(4.) D-AP7 (NMDA receptor antagonist) and NBQX (a non-NMDA receptor antagonist) did not alter the responses of any of the mGLUR agonists. The data presented may be useful in assessing the role of metabotropic and ionotropic GLURs in mediating different cardiovascular reflexes.

Cardiovascular responses to microinjections of nociceptin into a midline area in the commissural subnucleus of the nucleus tractus solitarius of the rat.

Immunoreactivity for nociceptin, an endogenous ligand for the ORL1 opioid receptors, has been reported in the nucleus tractus solitarius (nTS). A midline area in the commissural subnucleus (nCom) of nTS is the site of peripheral chemoreceptor projections. This investigation was carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats, to study the cardiovascular effects of the activation of ORL1 receptors in a midline area of the nCom. Microinjections (30 nl) of nociceptin (0.15-0.62 mM) into the nCom elicited depressor and bradycardic responses. Prior microinjections of [N-Phe(1)]-nociceptin-(1-13)-NH(2) (4.5 mM), a specific antagonist for ORL1 opioid receptors, into the nCom blocked the effects of nociceptin (0.31 mM, the maximally effective concentration), but not endomorphin-2 (0.6 mM; an endogenous ligand for micro -opioid receptors). On of other hand, naloxone (0.125 mM; an antagonist for classical opioid receptors) did not block the effects of nociceptin, while it did block the effects of endomorphin-2. The blockade of nociceptin effects by [N-Phe(1)]-nociceptin-(1-13)-NH(2) and endomorphin-2 by naloxone, was not due to some nonspecific effects because the responses to L-Glu (5 mM) remained unaltered after the microinjection of these antagonists. These results indicate that activation of ORL1 receptors in the nCom may play a role in the regulation of cardiovascular function.

Overexpression of the dopamine D3 receptor in the rat dorsal striatum induces dyskinetic behaviors.

L-DOPA-induced dyskinesias (LID) are motor side effects associated with treatment of Parkinson's disease (PD). The etiology of LID is not clear; however, studies have shown that the dopamine D3 receptor is upregulated in the basal ganglia of mice, rats and non-human primate models of LID. It is not known if the upregulation of D3 receptor is a cause or result of LID. In this paper we tested the hypothesis that overexpression of the dopamine D3 receptor in dorsal striatum, in the absence of dopamine depletion, will elicit LID. Replication-deficient recombinant adeno-associated virus-2 expressing the D3 receptor or enhanced green fluorescent protein (EGFP) were stereotaxically injected, unilaterally, into the dorsal striatum of adult rats. Post-hoc immunohistochemical analysis revealed that ectopic expression of the D3 receptor was limited to neurons near the injection sites in the dorsal striatum. Following a 3-week recovery period, rats were administered saline, 6 mg/kg L-DOPA, 0.1 mg/kg PD128907 or 10 mg/kg ES609, i.p., and motor behaviors scored. Rats overexpressing the D3 receptor specifically exhibited contralateral axial abnormal involuntary movements (AIMs) following administration of L-DOPA and PD128907 but not saline or the novel agonist ES609. Daily injection of 6 mg/kg L-DOPA to the rats overexpressing the D3 receptor also caused increased vacuous chewing behavior. These results suggest that overexpression of the D3 receptor in the dorsal striatum results in the acute expression of agonist-induced axial AIMs and chronic L-DOPA-induced vacuous chewing behavior. Agonists such as ES609 might provide a novel therapeutic approach to treat dyskinesia.