Astrocytes Modulate Baroreflex Sensitivity at the Level of the Nucleus of the Solitary Tract.

Maintenance of cardiorespiratory homeostasis depends on autonomic reflexes controlled by neuronal circuits of the brainstem. The neurophysiology and neuroanatomy of these reflex pathways are well understood, however, the mechanisms and functional significance of autonomic circuit modulation by glial cells remain largely unknown. In the experiments conducted in male laboratory rats we show that astrocytes of the nucleus of the solitary tract (NTS), the brain area that receives and integrates sensory information from the heart and blood vessels, respond to incoming afferent inputs with [Ca2+]i elevations. Astroglial [Ca2+]i responses are triggered by transmitters released by vagal afferents, glutamate acting at AMPA receptors and 5-HT acting at 5-HT2A receptors. In conscious freely behaving animals blockade of Ca2+-dependent vesicular release mechanisms in NTS astrocytes by virally driven expression of a dominant-negative SNARE protein (dnSNARE) increased baroreflex sensitivity by 70% (p < 0.001). This effect of compromised astroglial function was specific to the NTS as expression of dnSNARE in astrocytes of the ventrolateral brainstem had no effect. ATP is considered the principle gliotransmitter and is released by vesicular mechanisms blocked by dnSNARE expression. Consistent with this hypothesis, in anesthetized rats, pharmacological activation of P2Y1 purinoceptors in the NTS decreased baroreflex gain by 40% (p = 0.031), whereas blockade of P2Y1 receptors increased baroreflex gain by 57% (p = 0.018). These results suggest that glutamate and 5-HT, released by NTS afferent terminals, trigger Ca2+-dependent astroglial release of ATP to modulate baroreflex sensitivity via P2Y1 receptors. These data add to the growing body of evidence supporting an active role of astrocytes in brain information processing.SIGNIFICANCE STATEMENT Cardiorespiratory reflexes maintain autonomic balance and ensure cardiovascular health. Impaired baroreflex may contribute to the development of cardiovascular disease and serves as a robust predictor of cardiovascular and all-cause mortality. The data obtained in this study suggest that astrocytes are integral components of the brainstem mechanisms that process afferent information and modulate baroreflex sensitivity via the release of ATP. Any condition associated with higher levels of "ambient" ATP in the NTS would be expected to decrease baroreflex gain by the mechanism described here. As ATP is the primary signaling molecule of glial cells (astrocytes, microglia), responding to metabolic stress and inflammatory stimuli, our study suggests a plausible mechanism of how the central component of the baroreflex is affected in pathological conditions.

Abnormal oxygen homeostasis in the nucleus tractus solitarii of the spontaneously hypertensive rat.

NEW FINDINGS: What is the central question of this study? Arterial hypertension is associated with impaired neurovascular coupling in the somatosensory cortex. Abnormalities in activity-dependent oxygen consumption in brainstem regions involved in the control of cardiovascular reflexes have not been explored previously. What is the main finding and its importance? Using fast-cyclic voltammetry, we found that changes in local tissue PO2 in the nucleus tractus solitarii induced by electrical stimulation of the vagus nerve are significantly impaired in spontaneously hypertensive rats. This is consistent with previous observations showing that brainstem hypoxia plays an important role in the pathogenesis of arterial hypertension. The effects of arterial hypertension on cerebral blood flow remain poorly understood. Haemodynamic responses within the somatosensory cortex have been shown to be impaired in the spontaneously hypertensive rat (SHR) model. However, it is unknown whether arterial hypertension affects oxygen homeostasis in vital brainstem areas that control cardiovascular reflexes. In this study, we assessed vagus nerve stimulation-induced changes in local tissue PO2 (PtO2) in the caudal nucleus tractus solitarii (cNTS) of SHRs and normotensive Wistar rats. Measurements of PtO2 were performed using a novel application of fast-cyclic voltammetry, which allows higher temporal resolution of O2 changes than traditional optical fluorescence techniques. Electrical stimulation of the central cut end of the vagus nerve (ESVN) caused profound reductions in arterial blood pressure along with biphasic changes in PtO2 in the cNTS, characterized by a rapid decrease in PtO2 ('initial dip') followed by a post-stimulus overshoot above baseline. The initial dip was found to be significantly smaller in SHRs compared with normotensive Wistar rats even after ganglionic blockade. The post-ESVN overshoot was similar in both groups but was reduced in Wistar rats after ganglionic blockade. In conclusion, neural activity-dependent changes in tissue oxygen in brainstem cardiovascular autonomic centres are significantly impaired in animals with arterial hypertension.

Raised arterial blood pressure in neurokinin-1 receptor-deficient mice (NK1R(-/-) ): evidence for a neural rather than a vascular mechanism.

NEW FINDINGS: What is the central question of this study? Does genetic ablation of neurokinin-1 receptors alter arterial blood pressure? What is the main finding and its importance? NK1R(-/-) mice have increased mean arterial blood pressure, but without a concomitant change in vascular reactivity. This finding suggests that neurokinin-1 receptors play a role in the neural regulation of blood pressure. Mice with functional ablation of the neurokinin-1 receptor gene, Tacr1, (NK1R(-/-) ) express behavioural abnormalities equivalent to those seen in attention deficit hyperactivity disorder (ADHD). An established model of ADHD is the spontaneously hypertensive rat, which exhibits high blood pressure owing to increased central sympathetic drive. In light of the evidence that the neurokinin-1 receptor (NK1R) also influences cardiovascular haemodynamics, we have investigated whether NK1R(-/-) mice exhibit raised blood pressure. Cardiovascular parameters were recorded for 24 h in conscious mice using radiotelemetry. Vascular function was assessed in mesenteric resistance arteries by wire myography. The NK1R(-/-) mice exhibited a higher blood pressure than wild-type animals throughout the 24 h period. Heart rate and locomotor activity in NK1R(-/-) mice were higher than in wild-type mice during the night period (active phase), consistent with an ADHD-like phenotype, but not during the day. Mesenteric and renal arteries from NK1R(-/-) mice exhibited normal vascular function; the responses to vasoconstrictors (U46619 and phenylephrine) and the endothelium-dependent vasodilator, acetylcholine, were not altered in these animals, suggesting that the NK1R does not regulate vascular tone. Analysis of heart rate variability revealed a higher low-frequency to high-frequency ratio in NK1R(-/-) mice, indicative of increased cardiac sympathetic activity. We propose that the raised blood pressure in NK1R(-/-) mice could be due to a neural mechanism rather than a change in vascular reactivity. Further studies are required to understand this mechanism and to establish whether a subgroup of ADHD patients with polymorphism of the equivalent (TACR1) gene are affected in a similar way.

Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT).

The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12-50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg(-1) ). However, decynium-22 (600 µg kg(-1) ), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not noradrenaline, whereas desipramine had the reverse action. The OCT3 inhibitor corticosterone (10 mg kg(-1) ) had no effect. Blockade of glutamate receptors with topical kynurenate (10-50 nm) reduced the vagally evoked 5-HT signal by 50%, indicating that this release was from at least two sources. It is concluded that vagally evoked 5-HT release is under the regulation of the high-capacity, low-affinity transporter PMAT, not the low-capacity, high-affinity transporter SERT. This is the first demonstration that PMAT may be playing a physiological role in the regulation of 5-HT transmission and this could indicate that 5-HT is acting, in part, as a volume transmitter within the NTS.

5-hydroxytryptamine-mediated neurotransmission modulates spontaneous and vagal-evoked glutamate release in the nucleus of the solitary tract effect of uptake blockade.

The effect of blockade of either 5-hydroxytryptamine (5-HT)/serotonin transporter (SERT) with citalopram or the organic cation transporter 3 (OCT3)/plasma membrane monoamine transporter (PMAT) with decynium-22 (D-22) on spontaneous and evoked release of 5-HT in the nucleus tractus solitarius (NTS) was investigated in rat brainstem slices treated with gabazine. 5-HT release was measured indirectly by changes in the frequency and amplitude of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) [in the presence of tetrodotoxin (TTX)] and evoked EPSCs. Blockade of 5-HT3 receptors with granisetron reduced, whereas the 5-HT3 agonist phenylbiguanide increased, the frequency of mEPSCs. 5-HT decreased mEPSC frequency at low concentrations and increased frequency at high concentrations. This inhibition was blocked by the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635), which was ineffective on its own, whereas the excitation was reversed by granisetron. The addition of citalopram or D-22 caused inhibition, which was prevented by 5-HT1A blockade. Thus, in the NTS, the spontaneous release of 5-HT is able to activate 5-HT3 receptors, but not 5-HT1A receptors, as the release in their vicinity is removed by uptake. The ineffectiveness of corticosterone suggests that the low-affinity, high-capacity transporter is PMAT, not OCT3. For evoked 5-HT release, only D-22 caused an increase in the amplitude of EPSCs, with a decrease in the paired pulse ratio, and increased the number of spontaneous EPSCs after 20-Hz stimulation. Thus, for the evoked release of 5-HT, the low-affinity, high-capacity transporter PMAT, but not 5-HT transporter (5-HTT)/SERT, is important in the regulation of changes in 5-HT extracellular concentration.

Guidance for the use and reporting of anaesthetic agents in BJP manuscripts involving work with animals.

Scientists who plan to publish in the British Journal of Pharmacology (BJP) should read this article before undertaking studies utilising anaesthetics in mammalian animals. This editorial identifies certain gaps in the reporting of details on the use of anaesthetics in animal research studies published in the BJP. The editorial also provides guidance, based upon current best practices, for performing in vivo experiments that require anaesthesia. In addition, mechanisms of action and physiological impact of specific anaesthetic agents are discussed. Our goal is to identify best practices and to provide guidance on the information required for manuscripts submitted to the BJP that involve the use of anaesthetic agents in studies with experimental animals.

5-hydroxytryptamine and cardiovascular regulation.

All 5-hydroxytryptamine (5-HT; serotonin) receptors, except the 5-HT6 type, have been shown to be involved in cardiovascular regulation. In the periphery, 5-HT is stored in platelets, but the physiological role of 5-HT in the regulation of vascular tone (as opposed to its role in coagulation) remains unclear. However, central 5-HT1A, 5-HT3 and 5-HT7 receptors do play a physiological part in the regulation of cardiovascular reflexes, controlling changes in parasympathetic (vagal) drive to the heart. These reflexes also affect activity in the sympathetic nervous system, which itself can be inhibited by central 5-HT(1A) receptors to cause falls in blood pressure and excited by 5-HT2 receptors to cause rises in blood pressure. The physiological role of these receptors in the central regulation of the sympathetic nervous system is unclear, although 5-HT2 receptors could be involved in the development of deoxycorticosterone-acetate-salt hypertension, which is probably related to their role in the control of vasopressin release.

Involvement of central 5-HT7 receptors in modulation of cardiovascular reflexes in awake rats.

This study evaluated the role of 5-HT7 receptors within the central nervous system in modulating cardiovascular responses to the activation of chemo-, baro- and cardiopulmonary reflexes and in the regulation of mean arterial pressure and heart rate, using intracisternal (i.c.) application of the selective 5-HT7 receptor antagonist SB-269970 in awake rats. Experiments were performed on male Wistar rats (300-320 g). At 4 days before the experiment, rats were anesthetized and placed in a stereotaxic frame implantation of a guide cannula in the direction of the cisterna magna to be used for microinjection of saline or SB-269970 (100 microg/kg). On the day before the experiments a femoral artery and vein were cannulated to record arterial pressure and heart rate and to inject drugs to activate cardiovascular reflexes, respectively. The chemo-, baro- and cardiopulmonary reflexes were activated in different experimental groups before and after i.c. injection of saline or SB-269970. The antagonism of 5-HT7 receptors reduced: (a) the pressor (50+/-4 vs. 19+/-9 mm Hg) and bradycardic (-247+/-13 vs. -69+/-27 bpm) responses to chemoreflex activation; (b) the fall in MAP (-54+/-4 vs. -20+/-6 mm Hg) and the bradycardia (-294+/-12 vs. -98+/-34 bpm) in response to cardiopulmonary reflex activation; and (c) the gain of the baroreflex (-2.3+/-0.1 to -0.9+/-0.2 bpm/mm Hg). Intracisternal application of SB-269970 increased significantly baseline MAP in those rats previously submitted to the activation of a cardiovascular reflex but in naïve rats produced no changes in the baseline MAP were observed. The fact that cardiovascular responses to all reflexes tested were attenuated by the antagonism of 5-HT7 receptors suggests that brainstem 5-HT7 receptors brainstem facilitate the processing of the autonomic responses to cardiovascular reflex activation and that a 5-HT-containing pathway to the brainstem provides a normalizing input during challenges produced by cardiovascular reflex activation which seems to be mediated by 5-HT7 receptors.

The role of central 5-hydroxytryptamine (5-HT, serotonin) receptors in the control of micturition.

At present the most investigated 5-HT receptor that has been shown to play a role in the control of micturition is the 5-HT(1A) receptor followed by 5-HT(7), 5-HT(2) and 5-HT(3) receptors. Most experiments focus on the control these receptors have on the parasympathetic outflow to the bladder and the somatic outflow to the external urethral sphincter (EUS) in the rat. Furthermore, 5-HT(1A) and 5-HT(7) receptors have been identified as having an excitatory physiological role in the control of bladder function. 5-HT(1A) receptors act, at least in the rat, at both a spinal (probably a heteroreceptor) and supraspinal (probably an autoreceptor) level, while 5-HT(7) receptors only act at a supraspinal level. Additionally, in the rat, 5-HT administered at a spinal or supraspinal site has an excitatory action, although earlier experiments have shown that activating 5-HT-containing brain areas causes inhibition of the bladder. Recent experiments have also indicated that blockade of the 5-HT(1A) receptor pathway shows rapid tolerance. However, no data exist for the development of tolerance for the 5-HT(7) receptor pathway. Neither receptor seems to play a role in the control of the urethra. Regarding 5-HT(2) receptors, activation of this receptor subtype inhibits micturition, and this inhibitory action may occur at a spinal, supraspinal or both levels. Although no physiological role for 5-HT(2C) receptors can yet be identified, 5-HT(2C) receptors have been implicated in the proposed supraspinal tonically active 5-HT(1A) autoreceptor (negative feedback) pathway. This proposition reconciles the data that central 5-HT-containing pathways are inhibitory to micturition, while 5-HT(1A) receptors, although inhibitory to adenylyl cyclase, have an excitatory function. This is because activation of 5-HT(1A) autoreceptors reduces the release of 5-HT thus reducing the activation of the 5-HT(2C) receptors, which are inhibitory in the control of micturition (disinhibition). Furthermore, 5-HT(2A) receptors in the rat and 5-HT(2C) receptors in the guinea pig cause activation of the EUS. In this respect, 5-ht(5A) receptors have also been identified in Onuf's nucleus, the site of somatic motoneurones controlling this sphincter. In the cat there is very little evidence to indicate that 5-HT receptors are involved in micturition except under pathological conditions in which activation of 5-HT(1A) receptors causes inhibition of micturition. Interestingly, under such conditions 5-HT(1A) receptors cause excitation of the EUS. Nevertheless, spinal 5HT(3) receptors have been implicated in the physiological control of micturition in the cat, but not yet in the rat. Overall, the data support the view that 5-HT receptors are important in the control of micturition. However, many more studies are required to fully understand these roles and why there are such species differences.

Identifying the Source of a Humoral Factor of Remote (Pre)Conditioning Cardioprotection.

Signalling pathways underlying the phenomenon of remote ischaemic preconditioning (RPc) cardioprotection are not completely understood. The existing evidence agrees that intact sensory innervation of the remote tissue/organ is required for the release into the systemic circulation of preconditioning factor(s) capable of protecting a transplanted or isolated heart. However, the source and molecular identities of these factors remain unknown. Since the efficacy of RPc cardioprotection is critically dependent upon vagal activity and muscarinic mechanisms, we hypothesized that the humoral RPc factor is produced by the internal organ(s), which receive rich parasympathetic innervation. In a rat model of myocardial ischaemia/reperfusion injury we determined the efficacy of limb RPc in establishing cardioprotection after denervation of various visceral organs by sectioning celiac, hepatic, anterior and posterior gastric branches of the vagus nerve. Electrical stimulation was applied to individually sectioned branches to determine whether enhanced vagal input to a particular target area is sufficient to establish cardioprotection. It was found that RPc cardioprotection is abolished in conditions of either total subdiaphragmatic vagotomy, gastric vagotomy or sectioning of the posterior gastric branch. The efficacy of RPc cardioprotection was preserved when hepatic, celiac or anterior gastric vagal branches were cut. In the absence of remote ischaemia/reperfusion, electrical stimulation of the posterior gastric branch reduced infarct size, mimicking the effect of RPc. These data suggest that the circulating factor (or factors) of RPc are produced and released into the systemic circulation by the visceral organ(s) innervated by the posterior gastric branch of the vagus nerve.

Effect of 5-HT depletion on cardiovascular vagal reflex sensitivity in awake and anesthetized rats.

Antagonism of central 5-HT1A and 5-HT7 receptors inhibits reflex-evoked vagal bradycardias indicating that 5-HT is released during these reflexes. The present experiments examined the effect of 5-HT depletion with para-chlorophenylalanine (p-CPA) on the cardiac vagal baroreflex and cardiopulmonary reflex in awake and anesthetized rats. Immunocytochemistry and neurochemical detection showed that p-CPA depleted the brainstem of 5-HT, but not of norepinephrine or dopamine. Depletion of 5-HT was associated with an increase in mean arterial pressure (MAP) in awake rats. This difference was abolished by anesthesia, which reduced MAP in both groups of animals. The baroreflex gain, whether calculated from the rise in pressure induced by phenylephrine or the fall in pressure evoked by sodium nitroprusside, was significantly attenuated in depleted rats compared to controls. This attenuation of the baroreflex gain was unaffected by subsequent anesthesia. 5-HT depletion also attenuated the cardiopulmonary reflex vagal bradycardias but this only reached statistical significance when the rats were anesthetized. The data support the view that 5-HT is released in the reflex activation of the cardiac vagal pathway.

Chronic treatment with mianserin prevents DOCA-salt hypertension in rats--evidence for the involvement of central 5-HT2 receptors.

Central 5-HT2A receptors have been implicated in central volume control by activating a central angiotensinergic pathway to cause the release of vasopressin. Interestingly, to induce DOCA-salt hypertension in rats vasopressin release is required. Thus the present experiments were carried out to determine whether continuous blockade of these receptors over 20 days, with the non-selective 5-HT2 receptor antagonist mianserin would prevent the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Mianserin, given i.c.v. 90 or 60 microg twice daily for 20 days prevented the development of hypertension in conscious rats receiving DOCA-salt but did not affect blood pressure in rats on salt alone. Further, the dose of 30 microg given i.c.v. twice daily had no effect nor did the vehicle, polyethylene glycol (PEG), on the development of the hypertension. Mianserin 90 microg twice daily i.c.v. was also shown to prevent the increase in fluid intake, urinary flow and sodium excretion caused by DOCA-salt treatment. These data indicate that this action of mianserin is not due to an intrinsic hypotensive action but an action which involves interference with the mechanism by which DOCA-salt treatment causes hypertension. Thus the data overall support the view that to induce hypertension with DOCA-salt a central 5-HT-containing pathway needs to be activated, which then activates 5-HT2 receptors to cause the release of vasopressin which has previously been shown to be responsible for the initiation of DOCA-salt treatment hypertension.

Modulation of reflexly evoked vagal bradycardias by central 5-HT1A receptors in anaesthetized rabbits.

1. The role of central 5-HT(1A) receptors in the control of the bradycardia and changes in central respiratory drive, renal nerve activity and blood pressure evoked by stimulating cardiopulmonary afferents with phenylbiguanide, baroreceptors by electrical stimulation of the aortic nerve and chemoreceptors by injections of sodium cyanide (NaCN) in atenolol-pretreated anaesthetized rabbits were studied. 2. Buspirone (100 micro g kg(-1); i.c.) potentiated the bradycardia (increase in R-R interval) and the changes in blood pressure and renal nerve activity evoked by all three reflexes. These effects could be attenuated by pretreatment with the 5-HT(1A) receptor antagonist WAY-100635 (100 micro g kg(-1)); i.v.), which alone had no effect on these reflex-evoked changes. However, WAY-100635 (100 micro g kg(-1); i.c.) did attenuate these reflex-evoked responses produced by activation of cardiopulmonary and aortic baroreceptors but not that caused by stimulation of chemoreceptors. When given i.v., buspirone was less effective in modulating the responses evoked by these three reflexes. 3. The present data are consistent with the view that central 5-HT(1A) receptors play a role in the reflex activation of cardiac preganglionic vagal motoneurones. However, although antagonists of 5-HT(1A) receptors affected the responses evoked by cardiopulmonary and aortic nerve afferents, they were not effective on chemoreceptor reflex-evoked changes. This suggests that 5-HT(1A) receptors play a different role in chemoreceptor pathways compared to that for the other reflexes. This may relate to the fact that the chemoreceptor afferents travel in the IXth (glossopharyngeal) nerve whilst the other afferents travel in the Xth (vagus) nerve and thus may use different central circuitry and neurotransmitters.

An investigation of the effects of zaprinast, a PDE inhibitor, on the nitrergic control of the urethra in anaesthetized female rats.

1. The effects of L-NAME and zaprinast were investigated (i.v.) on reflex-evoked changes in bladder and urethral pressures in urethane-anaesthetized female rats. 2. L-NAME attenuated reflex-evoked urethral relaxations (65+/-10%), while zaprinast potentiated these responses (68+/-24%). L-NAME and zaprinast also increased baseline urethral pressure and urethral striated muscle (EUS-EMG) activity. These drugs had little effect on the bladder. 3. Following pre-treatment with alpha-bungarotoxin (i.v.) to block urethral striated muscle, L-NAME and zaprinast failed to increase baseline urethral pressure. Further zaprinast failed to alter the size of reflex-evoked urethral relaxations. 4. Intra-urethral zaprinast caused a significant increase while sodium nitroprusside (SNP) and isoprenaline caused decreases in urethral pressure (+14+/-3%, -25+/-5%, -29+/-7%, respectively). These changes were associated with increases in EUS-EMG activity. After chlorisondamine (i.v.), zaprinast caused a significant fall in urethral pressure, while the decrease in urethral pressure caused by SNP and isoprenaline was potentiated. No changes in EUS-EMG activity occurred. 5. These results indicate that a nitrergic pathway mediates reflex-evoked urethral smooth muscle relaxations. The data also indicates that there is a background release of NO, which reduces sphincter skeletal muscle activity. Further, the ability of zaprinast to potentiate nitrergic evoked urethral relaxations involves an increase in striated muscle tone. This appears to be an indirect result of smooth muscle relaxation and is mediated, at least in part, by a chlorisondamine-sensitive mechanism.

Investigation of the effects of P2 purinoceptor ligands on the micturition reflex in female urethane-anaesthetized rats.

1 The effects of purinoceptor ligands for P2X1 and/or P2X3 receptors (alpha,beta-meATP, IP(5)I, TNP-ATP, MRS 2179, PPADS, Phenol red and RO116-6446/008; i.v., n=4-5) and for P2Y1 receptors (PPADS, MRS 2179 and MRS 2269; i.v., n=3-5) were investigated on the distension-evoked 'micturition reflex' in the urethane-anaesthetized female rat. 2 Alpha,beta-meATP (180 nmol kg(-1) min(-1)), IP5I (10, 30 and 100 nmol kg(-1)), TNP-ATP (1 micromol kg(-1)), MRS 2179 (1 micromol kg(-1)) and PPADS (17 micromol kg(-1)) each caused maintained bladder contractions to occur during the infusion of saline into the bladder. PPADS (17 micromol kg(-1) min(-1)) had a similar effect when infused intravesicularly. Regular bladder contractions were not observed until the infusion of saline was halted. For IP5I, TNP-ATP, MRS 2179 and PPADS, the magnitude of postinfusion isovolumetric contractions was significantly reduced and, for IP5I, this action was also associated with a significant reduction in urethral relaxation. Additionally, TNP-ATP caused a significant increase in the pressure and volume thresholds required to initiate a reflex. 3 Phenol red (a P2X1/P2X3 antagonist; 0.1 and 1 micromol kg(-1)) caused a significant increase in the pressure and volume thresholds required to initiate a reflex and, at the higher dose, also caused a reduction in postinfusion isovolumetric contractions. 4 RO116-6446/008 (a P2X1-selective antagonist; 1 and 10 micromol kg(-1)) only caused a reduction in postinfusion isovolumetric contractions. 5 It is concluded that P2X1 and P2X3 receptors play a fundamental role in the micturition reflex in urethane-anesthetized female rats. P2X3 receptor blockade raised the pressure and volume thresholds for the reflex, whereas P2X1 receptor blockade diminished motor activity associated with voiding. P2Y1 receptors may be involved in inhibition of rat detrusor tone.

Evidence for the involvement of central 5-HT7 receptors in the micturition reflex in anaesthetized female rats.

(1) The effects of the selective 5-HT7 receptor antagonists SB-269970 (3-300 microg kg-1; n=5-6) and SB-656104 (30 microg kg-1; n=5) administered centrally (i.c.v.) were investigated on the 'micturition reflex' in the urethane anaesthetized female rat. (2) In cystometric recordings, SB-269970 caused significant increases in volume of 58+/-15 and 138+/-33% and pressure of 140+/-46 and 149+/-60% thresholds at 10 and 30 microg kg-1. These changes were associated with significant decreases in distension-induced bladder contraction of 62+/-14 and 60+/-11%, respectively. However, there was no change in residual volume. At the higher doses, SB-269970 blocked the micturition reflex. SB-656104 had similar effects to SB-269970 but in addition significantly increased the residual volume. (3) SB-269970 (10 microg kg-1; n=5) given i.v. had no effect on the micturition reflex. (4) SB-269970 (30 microg kg-1; n=4) given intrathecally (i.t.) had no effect on micturition reflex, although the selective 5-HT1A receptor antagonist WAY-100635 given i.t. after SB-269970 caused a significant increase in the volume threshold. (5) Using an isovolumetric method in which urethral changes were measured, SB-269970 (30 microg kg-1; n=4; i.c.v.) failed to have any effect on these urethral-evoked changes although they significantly reduced the amplitude of the bladder contraction. (6) These data demonstrate that 5-HT7 receptors located supraspinally in the rat are involved in the control of micturition.

Pharmacological studies of 8-OH-DPAT-induced pupillary dilation in anesthetized rats.

Serotonin (5-HT)(1A) receptor agonists have been reported to produce mydriasis in mice, and miosis in rabbits and humans. However, the underlying mechanisms for this action are unclear. This study was undertaken in an attempt to explore the mechanism by which 5-HT(1A) receptors are involved in the modulation of pupillary size in pentobarbital-anesthetized rats. Intravenous administration of the 5-HT(1A) receptor agonist, (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.003-3 mg/kg), elicited dose-dependent pupillary dilation, which was not affected by section of the preganglionic cervical sympathetic nerve. 8-OH-DPAT-elicited mydriatic responses were attenuated by the selective 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100635; 0.3-1 mg/kg, i.v.), as well as by the selective alpha(2)-adrenoceptor antagonist, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine hydrochloride (RS 79948; 0.3 mg/kg, i.v.), but not by the selective alpha(1)-adrenoceptor antagonist, prazosin (0.3 mg/kg, i.v.). Mydriatic responses elicited by the alpha(2)-adrenoceptor agonist, guanabenz (0.003-0.3 mg/kg, i.v.), were not antagonized by WAY 100635 (0.3-1 mg/kg, i.v.). To determine whether central nervous system (CNS) 5-HT(1A) receptors, like alpha(2)-adrenoceptors, are involved in reflex mydriasis, voltage response curves of pupillary dilation were constructed by stimulation of the sciatic nerve in anesthetized rats. WAY 100635 (1 mg/kg, i.v.) did not antagonize the evoked reflex mydriasis, which, however, was blocked by RS 79948 (0.3 mg/kg, i.v.). Taken together, these results suggest that 8-OH-DPAT produces pupillary dilation in anesthetized rats by stimulating CNS 5-HT(1A) receptors, which in turn trigger the release of norepinephrine, presumably from the locus coeruleus. The latter reduces parasympathetic neuronal tone to the iris sphincter muscle by stimulation of postsynaptic alpha(2)-adrenoceptors within the Edinger-Westphal nucleus. Unlike alpha(2)-adrenoceptors, 5-HT(1A) receptors in the CNS do not mediate reflex mydriasis evoked by sciatic nerve stimulation.

5-HT 2A receptor activation of the external urethral sphincter and 5-HT 2C receptor inhibition of micturition: a study based on pharmacokinetics in the anaesthetized female rat.

Central and peripheral 5-hydroxytryptamine (5-HT) receptors play a critical role in the regulation of micturition. Bolus doses of 5-HT(2A/2C) receptor agonists have been shown to activate the external urethral sphincter (EUS) and to inhibit micturition. This study was designed to determine the contribution of these two 5-HT receptor subtypes to activation of the EUS and inhibition of micturition utilising pharmacokinetic knowledge to better control drug exposure. Recordings of urethral and bladder pressure, EUS-Electromyogram (EMG), the micturition reflex induced by bladder filling, blood pressure and heart rate were made in anaesthetized female rats. The effects of intravenous (i.v.) infusions of the 5-HT(2) receptor agonist (2S)-1-(6-chloro-5-fluoroindol-1-yl)propan-2-amine fumarate (Ro 60-0175) in the absence or presence of the selective 5-HT(2C) receptor antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yl)oxypyridin-3-yl]-2,3-dihydroindole-1-carboxamide dihydrochloride (SB 242084) or 5-HT(2A) receptor antagonist (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (MDL-100,907) were studied on these variables. Continuous infusion of increasing concentrations of Ro 60-0175 only evoked EUS-EMG activity at the highest concentration, which was blocked by co-infusion of MDL-100,907 but not SB 242084. Urethral pressure was unaffected by any drug infusion. Ro 60-0175 at the lowest concentration inhibited the micturition reflex but as the concentration increased this was reversed to facilitation. SB 242084 blocked the inhibition while MDL-100,907 blocked the excitation. Activation of 5-HT(2A) not 5-HT(2C) receptors evoked EUS-EMG activity. In conclusion, 5-HT(2A) receptor activation facilitated the micturition reflex and evoked EUS-EMG while 5-HT(2C) receptor activation only inhibited the micturition reflex.

Nicotine's central cardiovascular actions: receptor subtypes involved and their possible physiological role in anaesthetized rats.

Centrally applied nicotine causes changes in blood pressure and vasopressin release. The involvement of different neuronal nicotinic receptor subtypes in these actions was investigated in anaesthetized rats. Nicotine administered i.c.v. caused a dose-related increase in blood pressure and renal sympathoinhibition, while i.c. administration also caused a bradycardia. In the presence of the V(1A) receptor antagonist (i.v.), nicotine (i.c) now caused a depressor response along with sympathoinhibition and bradycardia. Nicotine (0.3 µmol/kg; i.c.v.) in the presence of the α4ß2 receptor antagonist, dihydro-ß-erythroidine, (i.c.v.) evoked renal sympathoexcitation, while the α7 receptor antagonist, methyllycaconitine, delayed the expected sympathoinhibition. Both receptor antagonists blocked the pressor response. Dihydro-ß-erythroidine (i.c., 10µmol/kg) alone caused a transient pressor response and increased renal nerve activity. Methyllycaconitine (i.c., 0.1 µmol/kg) alone caused a slow fall in blood pressure and renal nerve activity, while the higher doses caused a pressor response and increased renal nerve activity. It was concluded that for nicotine to release vasopressin, activation of both α4ß2 and α7 receptors is required. The ability of nicotine to cause sympathoinhibition is mediated by ß4*-containing receptors, possibly α3ß4 receptors, and that activation of these receptors can override the sympathoexcitatory action of α4ß2 and α7 receptors. The ability of dihydro-ß-erythroidine and high doses of methyllycaconitine i.c. to cause sympathoexcitation and a pressor response is due to receptor antagonists blocking these sympathoinhibitory ß4*-containing receptors, which receive a tonic cholinergic input. As the low dose of methyllycaconitine caused sympathoinhibition, this indicates that sympathoexcitatory α7 receptors also receive a tonic input.

Both GABAA and GABAB receptors mediate vagal inhibition in nucleus tractus solitarii neurones in anaesthetized rats.

GABA receptors in the nucleus tractus solitarius (NTS) are known to play an important role in the mediation/modulation of various cardiovascular/respiratory functions. Vagal afferent activation of these neurones usually evokes an initial excitation followed by a long lasting inhibition. The present study examines the role of GABA(B) as well as GABA(A) receptors in the mediation of this inhibition in anaesthetized rats, using CGP 35348 and bicuculline, respective antagonists at these receptors, applied topically or by ionophoresis. Bicuculline delayed the onset and reduced the duration of this inhibition. The duration of this inhibition was further and significantly decreased when CGP 35348 was administered along with bicuculline but the delay in onset was unaffected. CGP 35348 application alone, had no effect on the vagal afferent-evoked inhibition. From intracellular recordings the early and late components of this inhibition were found to have reversal potentials close to E(Cl) and E(K), respectively. Therefore it is concluded that this inhibition is mediated by both GABA(A) and GABA(B) receptors, although GABA(B) mediated inhibition can only be unmasked when GABA(A) receptors are blocked.