Release of serotonin in the locus coeruleus of normotensive and spontaneously hypertensive rats (SHR)

The aim of the present work was to clarify whether differences exist between the release of endogenous serotonin in the locus coeruleus of normotensive and hypertensive rats. The locus coeruleus was superfused with artificial cerebrospinal fluid (aCSF) through a push-pull cannula and serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in the superfusate by HPLC combined with electrochemical detection. Compared with normotensive Wistar-Kyoto (WKY) rats, the basal release rate of serotonin in the locus coeruleus of spontaneously hypertensive rats (SHR) was increased more than twofold. Intravenous infusion of noradrenaline (4 μg/kg min) increased mean arterial blood pressure to the same extent in hypertensive and normotensive rats. The pressor response was associated with an increased serotonin release. In WKY rats, the release of serotonin in the locus coeruleus evoked by noradrenaline infusion was more pronounced than in SHR. In WKY rats, intravenous infusion of sodium nitroprusside (150 μg/kg min) led to a fall in blood pressure which was less pronounced and lasted shorter than in SHR. The depressor response was associated with decreased serotonin release. In WKY rats, the decrease in serotonin release evoked by sodium nitroprusside was more pronounced and lasted longer than in SHR. Neither noradrenaline nor sodium nitroprusside influenced the outflow of 5-HIAA. The sensory stimuli noise and tail pinch led to a slight rise in arterial blood pressure which was similar in WKY rats and SHR. These stimuli enhanced the release rate of serotonin and the outflow of 5-HIAA to the same extent in the locus coeruleus of normotensive and hypertensive rats. The findings suggest that the enhanced release of serotonin in the locus coeruleus of genetically hypertensive rats reflects a mechanism counteracting the disturbed blood pressure homeostasis. Stressors influence blood pressure and release of serotonin in the locus coeruleus of SHR and WKY rats to the same extent. Received: 16 November 1998 / Accepted: 22 February 1999     

Release of catecholamines in the locus coeruleus of freely moving and anaesthetized normotensive and spontaneously hypertensive rats: effects of cardiovascular changes and tail pinch

Noradrenaline turnover has been found to be increased in the locus coeruleus of young spontaneously hypertensive rats (SHR). There is also evidence that the noradrenergic projection from the locus coeruleus to the posterior hypothalamus contributes to the development of genetic hypertension. To investigate whether the release of noradrenaline and dopamine in the locus coeruleus is modified in genetic hypertension, this brain region of adult SHR and normotensive Wistar-Kyoto (WKY) rats was superfused with artificial cerebrospinal fluid through a push-pull cannula. Dopamine and noradrenaline released in the superfusate were determined radioenzymatically. There was no difference in the basal release of noradrenaline and dopamine in the locus coeruleus of conscious, anaesthetized or diazepam-treated adult WKY rats and SHR. In conscious animals, a rise in blood pressure elicited by intravenous infusion of phenylephrine enhanced the release of noradrenaline and dopamine in both strains to the same extent. Intravenous infusion of sodium nitroprusside elicited a fall in blood pressure and also increased to the same degree the release of noradrenaline and dopamine in the locus coeruleus of normotensive and hypertensive conscious rats. In anaesthetized rats, baroreceptor activation by phenylephrine decreased the release of noradrenaline and dopamine, while sodium nitroprusside lowered blood pressure and enhanced the release rates of the two catecholamines. Treatment of conscious rats with diazepam (10 mg/kg, i.p., 120 min prior to starting collection of the superfusate) abolished the phenylephrine-evoked release of catecholamines observed in conscious animals. The sensory stimulus tail pinch led to a slight increase in blood pressure. In conscious animals, this aversive stimulus led to enhanced release of noradrenaline and dopamine that lasted longer in SHR than in WKY rats. The release of catecholamines evoked by tail pinch was abolished in rats treated with diazepam, as well as in anaesthetized animals. Our findings show that in adult rats, genetic hypertension does not modify the release of noradrenaline and dopamine in the locus coeruleus. Since in anaesthetized rats increases in blood pressure diminish, while decreases in blood pressure enhance, the release of noradrenaline and dopamine, it seems that both amines possess a counteracting, hypertensive function in the rat locus coeruleus. When baroreceptor activation by phenylephrine is carried out on conscious animals, stress predominates and the release of catecholamines is enhanced. This study demonstrates the importance of the noradrenergic system of the locus coeruleus in central cardiovascular control and in emotional, stress and pain-regulating processes.     

Effects of inescapable shock and conditioned fear on the release of excitatory and inhibitory amino acids in the locus coeruleus

We investigated the importance of endogenous amino acids in the locus coeruleus in inescapable electric shock and conditioned fear. In naive rats and in rats exposed to noise (N), light (L) and electric shock (S) or to N + L only, the locus coeruleus was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of GABA, taurine, glutamate, aspartate, serine and glutamine was determined in the superfusate by HPLC after derivatization with o-phthaldialdehyde. Locomotor activity, arterial blood pressure and heart rate were telemetrically monitored. The placement of naive rats or conditioned rats from their home cage to a chamber provided with a grid-floor for shock virtually did not change the release rates of the amino acids in the locus coeruleus. Motility was enhanced in naive and conditioned rats to a similar extent. Blood pressure and heart rate were enhanced in conditioned rats only. Exposure to N + L + S for 5 min greatly enhanced the release rates of all determined amino acids in the locus coeruleus. In conditioned rats the increase in release of most amino acids lasted longer than in naive rats. Electric shock also enhanced motility, blood pressure and heart rate. In conditioned rats, motility and cardiovascular changes were more pronounced and/or lasted longer than in naive rats. Exposure of conditioned rats to the conditioned stimuli N + L for 5 min led to an increased release of taurine and aspartate. The enhanced release of taurine lasted 30 min. Exposure to N + L did not affect the release rates of amino acids in naive rats. N + L did not influence motility but arterial blood pressure and heart rate were elevated in conditioned rats. The findings show that inescapable electric shock enhances the release of several amino acids in the locus coeruleus, while conditioned fear selectively increases the outflow of taurine and aspartate. Moreover, conditioned fear prolongs the response of excitatory and inhibitory amino acids to electric shock. The results suggest that an excitatory amino acid (aspartate) and an inhibitory amino acid (taurine) of the locus coeruleus are implicated in conditioned fear.     

Dependence of serotonin release in the locus coeruleus on dorsal raphe neuronal activity

The serotonergic innervation of the locus coeruleus paetly derives from the dorsal raphe nucleus (DRN). Using the push-pull superfusion technique, we investigated whether and to what extent the release of serotonin and the extracellular concentration of its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the locus coeruleus are influenced by the neuronal activity of the DRN. In anaesthetized rats, a push-pull cannula was inserted into the locus coeruleus, which was continuously superfused with artificial cerebrospinal fluid (aCSF). Serotonin and 5-HIAA levels in the superfusate were determined by HPLC combined with electrochemical detection. Electrical stimulation (5 Hz, 300 μA, 1 ms) of the DRN for 5 min, or its chemical stimulation by microinjection of glutamate (3.5 nmol, 50 nl), led to an increased release of serotonin in the locus coeruleus and to a slight (2 mmHg) decrease in blood pressure. Superfusion of the locus coeruleus with tetrodotoxin (1 μM) abolished the increase in the release rate of serotonin evoked by electrical stimulation of the DRN, while the slight fall in blood pressure was not influenced. Thermic lesion (75 °C, 1 min) of the DRN elicited a pronounced decline in serotonin release rate within the locus coeruleus, the maximum decrease being 52%. The decrease in the release of serotonin was associated with a long-lasting rise in blood pressure. Microinjection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5 μg, 250 nl) into the DRN led to an initial increase in the serotonin release rate that coincided with a short-lasting fall in blood pressure. Subsequently, the release of serotonin was permanently reduced and was associated with hypertension. Microinjection of the 5-HT_1A receptor agonist (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 7.5 nmol, 50 nl) into the DRN led to a long-lasting reduction of the release rate of serotonin in the locus coeruleus. Microinjection of 8-OH-DPAT into the DRN also slightly lowered blood pressure (3 mmHg). Neither stimulations nor lesion of the DRN, nor microinjection of 8-OH-DPAT into this raphe nucleus, altered the extracellular concentration of 5-HIAA. Judging from the present biochemical results it appears that the serotonergic afferents to the locus coeruleus originate to more than 50% from cell bodies located in the DRN. The neuronal serotonin release in the locus coeruleus is modulated by 5-HT_1A receptors lying within the DRN. Changes in blood pressure and release of serotonin elicited by stimulating or lesioning the DRN point to the importance of serotonergic neurons extending between this raphe nucleus and the locus coeruleus in central cardiovascular control. Received: 5 November 1998 / Accepted: 21 February 1999     

Effect of vasoactive intestinal polypeptide on the release of serotonin from the in vitro vascularly perfused small intestine of guinea pig

Summary Isolated segments of the guinea pig small intestine were vascularly perfused and the release of endogenous serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) into the portal vein was measured. All test substances were intraarterially perfused. Vasoactive intestinal polypeptide (VIP, 1 pmol/l — 100 nmol/1) inhibited the spontaneous release of 5-HT and 5-HIAA. The maximal inhibitory effect (about 60%) was seen at 100 pmol/1. The effect of VIP on the spontaneous release of 5-HT and 5-HIAA was not changed in the presence of 1 ol/l tetrodotoxin (TTX).Raising intraluminal pressure by 500 Pa for 5 min increased the release of 5-HT and 5-HIAA by about 25%. Raising the intraluminal pressure in the presence of VIP reduced the release of 5-HT and 5-HIAA by about 75%. In the presence of TTX (1 gmol/l), raising intraluminal pressure also caused a decrease of the release of 5-HT and 5-HIAA which was unaffected by the additional presence of VIP. The fluid volume expelled during peristaltic activity was not affected by VIP, but reduced by about 90% in the presence of TTX.In conclusion the results demonstrate a direct inhibitory effect of VIP on the release of 5-HT from the enterochromaffin cells. In addition, VIP appears to interfere with the neuronally mediated stimulation of 5-HT release during peristaltic activity. Send offprint request to H. Schwörer at the above address     

The release of catecholamines in hypothalamus and locus coeruleus is modulated by peripheral chemoreceptors

To investigate whether impulses from chemoreceptors influence the release of catecholamines in the hypothalamus and the locus coeruleus, the two brain areas were superfused simultaneously and bilaterally with artificial cerebrospinal fluid through push-pull cannulae. The release of catecholamines was determined in the superfusate before and during chemoreceptor stimulation by bicarbonate solution saturated with carbon dioxide (CO2-NaHCO3) or KCN. Experiments were carried out on intact cats after carotid body denervation (CD). Intracarotid infusion of CO2-NaHCO3 increased arterial blood pressure and enhanced the release of noradrenaline but not dopamine in the posterior hypothalamus and the locus coeruleus. Following CD, the enhancing effect of CO2-NaHCO3 on the noradrenaline release in the posterior hypothalamus was abolished, while the effect on blood pressure was slightly enhanced. CD reversed the NaHCO3-induced release of noradrenaline in the locus coeruleus to a decreased noradrenaline outflow. Intracarotid infusion of KCN led to a fall in blood pressure. KCN increased the release rates of noradrenaline and, to a lesser extent, that of dopamine in the posterior hypothalamus, as well as the release of noradrenaline in the locus coeruleus. CD abolished the KCN-induced fall of blood pressure and the increased release of noradrenaline and dopamine in the posterior hypothalamus. Similar to CO2-NaHCO3, the enhancing effect of KCN on the noradrenaline release in the locus coeruleus was reversed following CD to a reduced noradrenaline outflow. Superfusion of the posterior hypothalamus and the locus coeruleus with KCN did not influence either blood pressure or the release rates of noradrenaline and dopamine in these brain areas. The findings show that impulses originating from chemoreceptors of the carotid body increase the release rates of the catecholamines in the posterior hypothalamus and the locus coeruleus, thus underlining the importance of catecholaminergic neurons of these brain areas in cardiovascular control.     

Inhibition of catecholamine (noradrenaline,dopamine) release in the locus coeruleus and the hypothalamus by baroreceptor activation: identification of the involved baroreceptors

We have previously shown that experimentally induced blood pressure changes modify the release rates of catecholamines in the hypothalamus and the locus coeruleus. The aim of the present investigation was to identify the peripheral baroreceptors and the centripetal pathways responsible for the changes of catecholamine release in these brain areas.In anaesthetized cats, push-pull cannulae were bilaterally inserted into the locus coeruleus and the posterior hypothalamus. The two brain areas were superfused simultaneously with artificial cerebrospinal fluid. Baroreceptor activation by phenylephrine-induced blood pressure elevation decreased the release rate of noradrenaline in the locus coeruleus and the release rates of noradrenaline and dopamine in the posterior hypothalamus. Similar effects were elicited by electrical stimulation of the central trunk of the transected vagus and aortic depressor nerves (vagus-ADN). Transection of the nerves abolished the effect of phenylephrine on the release of noradrenaline in the locus coeruleus. Nerve transections attenuated slightly the decreased release of noradrenaline elicited by phenylephrine in the posterior hypothalamus, while the reduced dopamine release rate was not influenced. The selective stimulation of baroreceptors in the carotid sinus by an inflatable catheter did not influence the release of catecholamines in the locus coeruleus, while release rates of noradrenaline and dopamine in the posterior hypothalamus were decreased.The simultaneous superfusion of locus coeruleus and hypothalamus revealed that, in both areas, noradrenaline release is inhibited by baroreceptor activation. Noradrenergic neurons of the posterior hypothalamus are inhibited by baroreceptor impulses conducted by the carotid sinus nerve and vagus-ADN, while the noradrenergic neurons of the locus coeruleus seem to respond to impulses transmitted by vagus-ADN. Furthermore, baroreceptor activation inhibits dopaminergic neurons in the hypothalamus but not in the locus coeruleus.     

Differential amino acid transmission in the locus coeruleus of Wistar Kyoto and spontaneously hypertensive rats

In addition to differences in their blood pressure, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) are known to differ in their emotional behaviour. The neurochemistry underlying these differences is not well understood. In the present study the release rates of the two main regulatory amino acids in the locus coeruleus, glutamate and gamma-aminobutyric acid (GABA), were monitored in WKY rats and SHR to investigate whether basal and/or challenged neurotransmission differs between these strains. The strains differed in their basal blood pressure (WKY 102±2 mmHg, SHR 140±4 mmHg), as well as in their emotional behaviour, since WKY rats displayed enhanced anxiety-related behaviour in the open field test (time in centre: WKY 197±40 s/30 min, SHR 741±93 s/30 min). Basal glutamate and GABA release rates did not differ between WKY rats and SHR. A rise in blood pressure induced by intravenous infusion of noradrenaline for 10 min enhanced GABA release in WKY rats by 60%, while no effect was observed in SHR. Glutamate release did not respond to experimental hypertension in both strains. Intravenous infusion of sodium nitroprusside led to a fall in blood pressure, which was less pronounced and was of shorter duration in WKY rats than in SHR. The depressor response had no effect on amino acid release in the locus coeruleus of both strains. Mild stress induced by noise or tail pinch led to slight rises in arterial blood pressure (10 mmHg and 20 mmHg respectively), which were similar in WKY rats and SHR. Tail pinch enhanced the release rates of glutamate and GABA in the locus coeruleus of WKY rats and SHR; however, no strain differences were noted. Noise stress did not significantly influence amino acid release. These findings demonstrate that SHR and WKY rats differ in GABAergic neurotransmission, which is revealed in response to specific cardiovascular challenges, but not to mild stressors. The observed lack of GABA response to blood pressure elevation in SHR may reflect a disturbed mechanism counteracting high blood pressure, possibly contributing to hypertension in this strain.     

Role of nitric oxide in the stress-induced release of serotonin in the locus coeruleus

Serotonergic mechanisms within the locus coeruleus (LC) are thought to be important in various functions including the stress response. In this study we investigated a possible role of nitric oxide (NO) as an intermediary messenger in the regulation of the serotonin (5-HT) neurotransmission within the LC. Using the push-pull superfusion technique coupled with HPLC and electrochemical detection, the in vivo release of 5-HT was determined in time periods of 10 min in the LC of freely moving rats. Superfusion with three different NO donors, SIN-1 (linsidomine), S-nitroso-N-penicillamine (SNAP) or 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPANO) increased 5-HT release in the LC. Superfusion with the precursor of NO, L-arginine, for 1 h led to a sustained increase in 5-HT release. On the other hand, the NOS inhibitor N-methyl-L-arginine methyl ester (L-NAME) did not significantly change the release of 5-HT. Infusion of N-methyl-D-aspartate (NMDA) or kainic acid, as well as exposure of rats to noise stress or tail pinch increased the release of 5-HT in the LC. Superfusion with L-NAME prevented the increase in 5-HT outflow by all these procedures, while the inactive isomer D-NAME had no effect. Taken together, the results of this study suggest that the release of 5-HT in the LC is facilitated by NO. Under resting conditions inhibition of NOS does not appear to substantially influence the release of 5-HT in the LC. However, there seems to be a facilitatory nitrergic influence on serotonergic responses evoked by excitatory amino acid receptor stimulation or various stress stimuli.     

Cholinergic modulation of the release of 5-hydroxytryptamine from the guinea pig ileum

Summary Isolated segements of the guinea pig ileum were vascularly perfused and the release of 5-HT and its metabolite 5-HIAA into the portal venous effluent determined by HPLC with electrochemical detection. Test substances were applied via the arterial perfusion medium. Oxotremorine inhibited concentration-dependently the release of 5-HT and 5-HIAA (by 47% at 1 mol/l). Scopolamine (0.1 mol/1) did not affect the release of 5-HT and 5-HIAA, but antagonized the effect of oxotremorine. In the presence of TTX (1 mol/1) oxotremorine (1 pmol/1) increased the release of 5-HT by 150% and that of 5-HIAA by 220%. This increase was completely blocked by scopolamine. Hexamethonium (100 pmol/1) and TTX (1 pmol/1) reduced the release of 5-HT by 32 and 40%, respectively. DMPP (10 pmol/1) increased the release of 5-HT by 57%, and this effect was prevented by hexamethonium. Neither DMPP nor hexamethonium significantly affected the release of 5-HIAA. The enhancing effect of DMPP on 5-HT release was increased and prolonged in the presence of TTX or scopolamine. Nicotine (1, 10 or 30 mol/l) alone did not cause a consistent increase in the release of 5-HT. However, in the presence of scopolamine nicotine increased the release of 5-HT by 57%. In conclusion, the release of intestinal 5-HT is facilitated via muscarine and nicotine receptors located on the enterochromaffin cells. Indirect evidence suggests that the release of 5-HT is additionally modulated by an as yet unknown inhibitory neurotransmitter released by muscarine receptor activation.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium - 5-HT 5-hydroxytryptamine - 5-HIAA 5-hydroxyindoleacetic acid - TTX tetrodotoxin Send offprint requests to H. S. at the above address     

Acute effect of milnacipran on the relationship between the locus coeruleus noradrenergic and dorsal raphe serotonergic neuronal transmitters

The present studies sought to investigate the effect of milnacipran called the serotonin (5-HT) and noradrenaline (NA) reuptake inhibitor (SNRI) on the interaction of central locus coeruleus noradrenergic and dorsal raphe nucleus serotonergic functional activity by utilizing in vivo microdialysis. A single administration of milnacipran (60 mg/kg, s.c.) markedly decreased the levels of NA and its metabolite, 4-hydroxy-3-methoxymandelic acid (HMMA), in the locus coeruleus and the levels of, a metabolite of 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) in the dorsal raphe nucleus. Combined administration of yohimbine (2 mg/kg, s.c.),?alpha(2)-adrenoceptor?antagonist, at 2 h after milnacipran (60 mg/kg, s.c.) led to a significant increase in NA levels in the locus coeruleus, although yohimbine alone had no effect on these levels. Under similar experimental condition, 5-HIAA levels in the dorsal raphe nucleus remained unchanged. NAN-190 (1 mg/kg, s.c.), 5-HT(1A) receptor partial agonist, alone markedly decreased the levels of 5-HIAA in the dorsal raphe nucleus, although this level was not affected by WAY100635, the selective 5-HT(1A) receptor antagonist. WAY100635 recovered the milnacipran-induced decrease of 5-HIAA levels in the dorsal raphe nucleus to control levels. On the other hand, NAN-190 did not affect the milnacipran-induced decrease of 5-HIAA levels. Behavioral signs (locomotion and rearing) were markedly observed following milnacipran alone or combined administration of milnacipran and yohimbine. However, the behavioral signs after coadministration of milnacipran and WAY100635 or NAN-190 were relatively poor. These results may suggest that an increase of NA in the locus coeruleus with the treatment of yohimbine after milnacipran results from negative feedback following the blockade of alpha(2)-adrenoceptors achieved with yohimbine, and that WAY100635 but not NAN-190 recovered milnacipran-induced decrease of 5-HIAA in the dorsal raphe nucleus to control levels by preventing the activation for the presynaptic 5-HT(1A) autoreceptor.     

Acute effect of simultaneous administration of tryptophan and ethanol on serotonin metabolites in the locus coeruleus in rats

Using the microdialysis method, we investigated whether the levels of serotonin (5-hydroxytryptamine, 5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTPL), in the locus coeruleus are influenced by tryptophan alone or simultaneous administration of tryptophan and ethanol. Tryptophan (50 mg/kg, i.p.) led to a significant increase in the levels of 5-HIAA, but not 5-HT in the locus coeruleus. However, ethanol (1.25 g/kg) had no effect on the levels of 5-HT and its metabolites. Combined administration of tryptophan and ethanol caused very marked increases in 5-HIAA and 5-HTPL levels in the locus coeruleus. A time lag in the increased 5-HIAA levels between tryptophan alone and tryptophan plus ethanol was observed. Moreover, 5-HIAA levels in the locus coeruleus induced by tryptophan were abolished by microinjection of 5,7-dihydroxytryptamine (150 microg/4 microl) into the dorsal raphe nucleus. Judging from the present results, the serotonergic afferents to the locus coeruleus may originate for about 20-30% from cell bodies located in the dorsal raphe nucleus. Teeth-chattering was significantly detected in the tryptophan plus ethanol-treated rats when compared with the tryptophan-treated rats, but not in the saline-treated controls. These results may suggest that the increased levels of 5-HIAA and 5-HTPL in the locus coeruleus induced by tryptophan are potentiated by ethanol, and that these levels are partly responsible for behavioral activation.     

Differential effects of electrical stimulation,blockade of neuronal amine uptake and activation of α2-adrenoceptors on the release of endogenous noradrenaline and 5-hydroxytryptamine from the isolated rat pineal gland

Summary Isolated rat pineal glands were incubated in vitro and the release of endogenous noradrenaline or 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection. In the absence of test drugs, the spontaneous outflow of noradrenaline was about 10 fmol/10 min and electrical stimulation (5 Hz, 1500 pulses) evoked the release of about 70 fmol noradrenaline. Nomifensine enhanced the spontaneous outflow of noradrenaline about threefold and the electrically evoked release of noradrenaline about sixfold. In the presence of nomifensine, the ₂-adrenoceptor antagonist yohimbine markedly increased the electrically evoked release of noradrenaline, whereas the ₁-adrenoceptor antagonist prazosin had no effect. Clonidine inhibited the electrically evoked release of noradrenaline by about 65%, and this was antagonized by yohimbine in a competitive manner. In the absence of drugs, the initial spontaneous outflow of 5-HT was (compared with noradrenaline) very high 64 mol/10 min. It declined by 80% within 1 h of incubation in vitro. The outflow of 5-HIAA amounted initially to 38 mol/10 min and declined by 40% within 1 h of incubation. Addition of l-tryptophan (10 mol/1) after 1 h of incubation in vitro largely enhanced the outflow of 5-HT and 5-HIAA within 30 min of incubation (about ten- and fourfold, respectively). When l-tryptophan was present from the onset of incubation the initial outflow of 5-HT and 5-HIAA was only slightly elevated, but the decline was largely attenuated. Neither omission of calcium nor addition of nomifensine, clonidine or yohimbine significantly affected the spontaneous outflow of 5-HT or 5-HIAA. Likewise, neither electrical stimulation in the absence or presence of nomifensine and yohimbine nor stimulation by high potassium (45 mmol/1) significantly affected the outflow of 5-HT or 5-HIAA.In conclusion, the release of endogenous noradrenaline from the sympathetic nerves terminating in the pin eal gland is inhibited by presynaptic ₂-adrenoceptors. The outflow of 5-HT from the pineal gland originates almost exclusively from non-neuronal cells, most probably the pinealocytes, and depends largely on a continuous de novo synthesis. Catabolism of 5-HT to 5-HIAA in the pineal gland occurs mainly in an extraneuronal compartment, probably the pinealocytes and/or the interstitial cells of the pineal gland. Send offprint requests to K. Racké at the above address     

Effects of blood pressure changes on the catecholamine release in the locus coeruleus of cats anaesthetized with pentobarbital or chloralose

Summary Effects of carotid occlusion and drugs applied intravenously on the release of endogenous catecholamines in the locus coeruleus of cats anaesthetized with pentobarbital or chloralose were investigated. The locus coeruleus was superfused bilaterally with artificial cerebrospinal fluid through push-pull cannulae inserted stereotaxically. Dopamine, noradrenaline and in some experiments also adrenaline were determined radioenzymatically in the superfusate.Under pentobarbital anaesthesia, a bilateral carotid occlusion increased the release rate of noradrenaline in the locus coeruleus, while the release of dopamine was decreased. These changes were due to the fall of blood pressure in the carotid sinus caused by the occlusion. Loading of baroreceptors by elevating blood pressure with phenylephrine (10 g·kg^–1·min^–1, i.v. infusion) was accompanied by a decreased release of noradrenaline in the locus coeruleus. This decrease in noradrenaline release was not detected in the caudal aspect of the locus coeruleus. Under chloralose anaesthesia, phenylephrine diminished the release rate of noradrenaline to about the same extent as under pentobarbital anaesthesia. The release rate of adrenaline was also decreased. A prolonged infusion of phenylephrine led to a prolonged pressor response associated with a sustained decrease in the noradrenaline release rate. Intravenous injection of chlorisondamine (3 mg·kg^–1) did not change the release of noradrenaline, while dopamine release was reduced.It is concluded that the release of catecholamines in the locus coeruleus is influenced by signals originating from peripheral baroreceptors. The influences are similar under pentobarbital and chloralose anaesthesia. Noradrenergic neurons responding to haemodynamic signals are not uniformly distributed within the locus coeruleus. It is suggested that noradrenergic and possibly dopaminergic and adrenergic neurons of the locus coeruleus are involved in the baroreceptor reflex, thus contributing to central homeostasis of blood pressure.     

Endogenous serotonin enhances the release of dopamine in the striatum only when nigro-striatal dopaminergic transmission is activated

In this study, we use in vivo microdialysis to investigate the influence of endogenous serotonin (5-HT) on striatal dopamine (DA) and 5-hydroxyidoleacetic acid (5-HIAA) efflux in both basal and activated conditions. The selective serotonin reuptake inhibitors citalopram and fluoxetine were used to mobilize endogenous 5-HT.In halothane-anaesthetized rats, citalopram (5 mg/kg, i.p.), administered either alone or in combination with the 5-HT(1A) receptor antagonist WAY 100635 (0.1 mg/kg, s.c.), while reducing striatal 5-HIAA outflow (-25 and -15%, respectively), had no effect on basal DA output. When locally applied into the striatum, citalopram had no effect at 1 microM concentration, but enhanced DA release after its perfusion at 25 and 100 mircroM concentrations (+27% and +67%, respectively). However, the injection of the neurotoxin 5,7-dihydroxytryptamine into the dorsal raphe nucleus, which markedly depleted 5-HT in the striatum, failed to modify the effect of 25 microM citalopram.In freely-moving rats, the intrastriatal infusion of citalopram or fluoxetine (1 microM each), had no effect on its own, but significantly enhanced the increase in DA outflow induced by the subcutaneous administration of 0.01 mg/kg haloperidol (+31% and +30% for citalopram and fluoxetine, respectively).These findings indicate that, in the striatum, endogenous 5-HT has no influence on DA release under basal conditions, but positively modulates DA outflow when nigro-striatal DA transmission is activated.     

利血平和帕吉林对大鼠脑单胺递质含量的联合作用

大鼠ip利血平2mg/kg后1-24h,NE,DA,5-HT均显著下降(p<0.05-p<0.001),5-HIAA则显著升高(p<0.001)。ip帕吉林60mg/kg后1-24h,NE,DA,5-HT均显著升高(p<0.01-p<0.001),5-HIAA则在1-2h内显著下降(p<0.01),4-24h无明显变化。同时使用两药时,对鼠脑NE和DA的影响均呈拮抗作用,对5-HT仅表现帕吉林的作用。帕吉林对利血平化鼠的NE,DA无影响,但可使5-HT升至正常水平;利血平对帕吉林化鼠递质的变化无影响。两药合用对递质的影响表现为拮抗或单一作用,未见递质翻转现象。     

The interaction between the locus coeruleus and dorsal raphe nucleus studied with dual-probe microdialysis

The interaction between the locus coeruleus and dorsal raphe nucleus was investigated by means of dual-probe microdialysis in conscious rats. The release of noradrenaline and 5-hydroxytryptamine (5-HT) after inhibition or stimulation of locus coeruleus and dorsal raphe activity was sampled in both nuclei and analysed by high-pressure liquid chromatography (HPLC). The inhibition of locus coeruleus activity by the infusion of the alpha(2)-adrenoceptor agonist clonidine (100 microM) decreased the release of noradrenaline to 20% in the locus coeruleus and 30% in the dorsal raphe, whilst the release of 5-HT decreased to 80% of control in the two brain areas. The excitation of locus coeruleus activity by the muscarinic receptor agonist carbachol (100 microM) led to an increase in the release of noradrenaline to 240% and 220% of control in the locus coeruleus and dorsal raphe, respectively. The release of 5-HT in both nuclei did not respond to the carbachol infusion into the locus coeruleus. Infusion of the 5-HT(1A) receptor agonist flesinoxan into the dorsal raphe (1 microM) significantly decreased the release of 5-HT in the dorsal raphe and locus coeruleus to 45% and 65% of control, respectively. The release of noradrenaline was decreased in the dorsal raphe to 45% by flesinoxan, whereas no changes were seen in the release of noradrenaline in the locus coeruleus. In conclusion, the innervation of the dorsal raphe by the locus coeruleus has a slight excitatory effect on the release of 5-HT in the dorsal raphe. The dorsal raphe does not exert a direct inhibitory influence on the release of noradrenaline in the locus coeruleus. Finally, the release of noradrenaline in the dorsal raphe may be locally regulated by 5-HT(1A) receptors.     

In vivo release of endogenous catecholamines in the hypothalamus

Summary The posterior hypothalamus of anaesthetized cats was superfused with a push-pull cannula and the release of the endogenous catecholamines noradrenaline, adrenaline and dopamine was determined in the superfusate. The rate of release of the three catecholamines followed an ultradian rhythm, the time interval between two adjacent phases of high rate of release being about 70 min. Pretreatment of the animals with reserpine decreased the levels of catecholamines in the hypothalamus and rest of the brain and reduced their rate of release into the superfusate. Hypothalamic superfusion with superfusing fluid of high concentration of potassium and low concentration of sodium enhanced the rate of release of noradrenaline and adrenaline; this effect was abolished when the hypothalamus was superfused with calcium-free solution. Electrical stimulation of the locus coeruleus ipsilateral to the superfused hypothalamus increased the release of noradrenaline and adrenaline, stimulation of the contralateral locus coeruleus enhanced the release of noradrenaline, adrenaline and dopamine. In both cases, the rate of release of adrenaline was enhanced to a lesser extent than the rate of release of noradrenaline. The release of noradrenaline and adrenaline was increased to a higher extent on stimulation of the ipsilateral locus coeruleus than on stimulation of the contralateral one.Part of the results was presented at the Spring Meeting of the German Pharmacological Society, Mainz, March 1978 and at the IV International Catecholamine Symposium, Asilomar, September 1978This work was supported by the Deutsche Forschungsgemeinschaft     

Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: Involvement of 5-HT3 receptors

Summary Isolated segments of the guinea-pig small intestine were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Release of acetylcholine from isolated superfused intestinal segments was determined as outflow of [³H]radioactivity from preparations preincubated with [³H]choline. Cisplatin (3 M) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 M cisplatin decreased the outflow of 5-HT and its metabolite by 40%–50%. The stimulatory effect of cisplatin was consistently observed only when the bicarbonate-phosphate buffer of the Tyrode's solution was replaced by HEPES-buffer. The stimulatory effect of cisplatin was abolished in the absence of extracellular calcium or presence of tetrodotoxin (1 M). The stimulatory effect of cisplatin was also prevented by hexamethonium (100 M) or scopolamine (100 nM). The 5-HT₃ receptor antagonists ondansetron and ICS 205-930 in concentrations as low as 1 pM also abolished the stimulatory effect of cisplatin. The 5-HT₃ receptor antagonist MDL 72222 prevented the stimulatory effect of cisplatin only at a concentration of 1 M. None of the 5-HT₃ receptor antagonists alone significantly altered the outflow of 5-HT and 5-HIAA.Cisplatin (3 M) enhanced the outflow of [³H]radioactivity from intestinal segments and caused longitudinal muscle contractions that were abolished by 100 nM scopolamine.In conclusion, cisplatin, at concentrations which occur during anti-cancer therapy in humans and induce emesis, increases the release of 5-HT from the enterochromaffin cells of the small intestine of the guinea-pig. This effect of cisplatin is mediated by a cascade of events which involves release of acetylcholine and stimulation of 5-HT₃ receptors. Send offprint requests to H. Schwörer at his present address     

In-vivo Microdialysis Measurement of 5-Hydroxytryptamine and its Metabolites, 5-Hydroxyindoleacetic Acid and N-Acetyl 5-Hydroxytryptamine,in Rat Blood: Effects of Histamine-receptor Antagonists

The blood concentrations of 5-hydroxytryptamine (5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and N-acetyl 5-HT were assayed by in-vivo microdialysis and a highly sensitive HPLC procedure that was originally developed to analyse CNS mediators. We investigated the effects of histamine-receptor antagonists on 5-HT metabolism and its release into the blood of rats. The mean basal levels of 5-HT, 5-HIAA and N-acetyl 5-HT in the blood measured by in-vivo microdialysis were 77.2 ± 9.4, 20.3 ± 1.5 and 1.89 ± 0.15 pmol mL^?1, respectively. These levels were not significantly affected by an intraperitoneal injection of saline, and remained at constant levels for at least 8 h after administration of saline. After an intraperitoneal injection of 5-HT hydrochloride (0.5 mg kg^?1), 5-HT was soon detected in the blood of the jugular vein. 5-HIAA also quickly appeared in the blood and declined monoexponentially from 60 min after injection. In contrast, N-acetyl 5-HT slowly appeared in the blood and it reached a maximal level at 270 min. The 5-HT and N-aceryl 5-HT levels in dialysates from rat jugular vein were significantly increased by intraperitoneal pyrilamine (2.0 mg kg^?1), (+)-chlorpheniramine (2.0 mg kg^?1) and cimetidine (20.0 mg kg^?1). However, there was no increase in the 5-HIAA concentration after an intraperitoneal injection of these histamine-receptor antagonists, demonstrating that the 5-HT released from various cells containing 5-HT was predominantly metabolized to N-acetyl 5-HT by N-acetyltransferase. Moreover, thioperamide did not affect the basal levels of 5-HT, 5-HIAA or N-acetyl 5-HT. Because the recovered 5-HT, 5-HIAA and N-acetyl 5-HT in the dialysate is directly proportional to the free fraction in the blood, in-vivo microdialysis is a reliable method of examining 5-HT metabolism and its release into the blood.