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Peripheral chemoreceptor activation enhances 5-hydroxytryptamine release in the locus coeruleus of conscious rats
Neuroscience Letters 289 (2000) 17±20
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Peripheral chemoreceptor activation enhances 5-hydroxytryptamine release in the locus coeruleus of conscious rats Nicolas Singewald a,*, Dimitrios Kouvelas b, Stefan T. Kaehler a, Catrin Sinner a, Athineos Philippu a a
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, Peter-Mayr-Straûe 1, A-6020 Innsbruck, Austria b Department of Pharmacology, Aristotle University of Thessaloniki, Greece Received 10 April 2000; received in revised form 31 May 2000; accepted 31 May 2000
Abstract Intravenous bolus injection of KCN (40 mg) elicited brief but pronounced tachypnea, bradycardia and pressor response, and led to a 37% increase in 5-hydroxytryptamine (serotonin) (5-HT) release in the locus coeruleus (LC) of freely moving rats. Slow infusion of KCN (15 mg/min) for 10 min induced only a slight pressor response, but increased the respiration rate (139 breaths/min), as well as 5-HT release in the LC (160%) throughout the infusion. In rats with transected chemoreceptor afferents, neither injection or infusion of KCN changed 5-HT release, suggesting that in intact animals, the effect on extracellular 5-HT was due to activation of peripheral chemoreceptors. In summary, we report that peripheral chemoreceptor activation enhances 5-HT release in the LC, indicating that 5-HT might be involved in the modulation of LC activity by ascending chemosensory information. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 5-Hydroxytryptamine; Push-pull superfusion; Sinoaortic denervation; Chemore¯ex; Respiration; Blood pressure; Cyanide
Chemoreceptor activation by brief periods of hypoxia and hypercapnia have been shown to enhance the discharge rate of locus coeruleus (LC) noradrenergic neurons in anaesthetized animals [2,5]. Peripheral chemoreceptor stimulation by potassium cyanide (KCN) or CO2-saturated NaHCO3 enhances the release of noradrenaline in the LC of anaesthetized cats [9]. Both, enhanced ®ring of LC neurons as well as enhanced noradrenaline release in response to brief hypoxia or KCN can be reversed by transection of chemoreceptor afferents [2,5,9], showing that the activation of the LC noradrenergic system by these stimuli is indeed produced by peripheral chemoreceptor stimulation. In conscious animals, hypoxia has been shown to enhance cfos expression in the LC [3,8]. Although these and other studies show that the LC noradrenergic system is responsive to chemosensory stimuli, the afferent pathways and neurotransmitters involved in this modulation of LC neurons are little investigated. * Corresponding author. Tel.: 143-512-507-5601; fax: 143-512507-2931. E-mail address: [email protected] (N. Singewald).
Using the push-pull superfusion technique, we investigated in the present study whether 5-HT release in the LC of freely moving rats is in¯uenced by chemoreceptor activation. For this purpose, KCN was applied intravenously as a model of peripheral chemoreceptor activation [4,6,7,21] and its effects on blood pressure (BP), respiration and neurotransmitter release in the LC were studied. We have focused our interest on 5-HT release, since the LC is densely innervated by serotonergic ®bers (for review see [15]) and 5-HT has been suggested to play a role in the control of respiration in other parts of the brain, in particular the medulla (for review see [3]). Male Sprague±Dawley rats (280±300 g) were anaesthetized with diethylether and ketamine 50 mg/kg (i.p.). A guide cannula was stereotaxically inserted with its tip 2 mm above the LC [16]. The iliac artery and jugular vein were catheterized for recording of arterial BP, heart rate and intravenous infusions of drugs, respectively. In one group of rats peripheral chemoreceptor denervation involving transection of carotid sinus and aortic depressor nerves (SAD) [4]
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 24 1- 6
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N. Singewald et al. / Neuroscience Letters 289 (2000) 17±20
was carried out. Sham operated rats were subjected to the same surgical procedure without transection of nerves. At least ®ve days after surgery, the stylet of the guide cannula was replaced by a push-pull cannula which was 2 mm longer than the guide cannula thus reaching the LC. The coordinates were (mm): Anterior-posterior (AP) 0.8 posterior to interaural line, L 1.3, dorsal-ventral (DV) 2.8 above the interaural zero plane [11]. The LC was superfused with arti®cial cerebrospinal ¯uid (aCSF) at a rate of 14 ml/min. Superfusate was continuously collected in time periods of 10 min. 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) was determined in the superfusate by high pressure liquid chromatography (HPLC) with electrochemical detection [16]. Localization of the push-pull cannula was veri®ed histologically. Data are expressed as percentage of controls values. The mean release rate of 5-HT and mean arterial BP in the three samples preceding KCN administration were taken as control values. Statistical analysis was carried out by Friedman's analysis of variance followed by Wilcoxon's signed rank test for paired data. The mean basal release of 5-HT in sham operated rats was 1.4 ^ 0.1 fmol/min (mean ^ SEM, n 19) which is in good accordance with 5-HT release rates in the LC of intact rats that have not received sham operation [16,17]. SAD rats displayed similar release rates (1.5 ^ 0.2 fmol/min, mean ^
Fig. 1. Effects of intravenous KCN bolus injection (40 mg) on 5HT release in the LC and arterial blood pressure (BP). Mean release rate and mean BP in the three samples preceding KCN injection were taken as 100%. Arrow indicates KCN bolus injection. SAD chemoreceptor denervation. Mean ^ SEM *P , 0:05.
SEM, n 10), indicating that transection of chemoreceptor afferents had no effect on basal 5-HT release in the LC. Also resting BP did not differ in sham-operated (116 ^ 5 mmHg, mean ^ SEM, n 19) and SAD rats (111 ^ 6 mmHg, mean ^ SEM, n 10). In sham operated rats, intravenous bolus injection of KCN (40 mg) elicited a pressor response (160 mmHg, 152% from basal), which peaked within 10 s and lasted for 20 s (Fig. 1). KCN injection also led to brief bradycardia (2126 ^ 12 beats/min) and enhanced the respiration rate (146 ^ 5 breaths/min) for 2 min (not shown). KCN bolus injection enhanced the release rate of 5-HT in the LC by 37% (Fig. 1), while 5-HIAA out¯ow was not in¯uenced (not shown). In SAD rats, the effects of KCN on 5-HT release and arterial BP were abolished (Fig. 1). The slow intravenous infusion of KCN (15 mg/min) for 10 min elicited only a slight pressor response in sham operated rats which lasted for 2.5 min (Fig. 2). Respiration rate was increased throughout the infusion with KCN by 39 ^ 5 breaths/min (mean change ^ SEM, not shown). 5-HT release was enhanced by 60% during KCN infusion and was immediately normalized after termination of the infusion (Fig. 2). Enhanced 5-HT release as well as the pressor response to KCN infusion were abolished in SAD rats. Infusion of a higher dose of KCN (30 mg/min) led to a prolonged (20 min) increase in 5-HT release and decreased arterial BP by 10±15 mmHg (Fig. 3). The hypotensive response to the high dose of KCN was similar in sham operated and SAD rats, while the increase in 5-HT was abolished in SAD rats. Intravenous infusion of noradrenaline (0.8±1 mg/kg per min, n 6) led to a pressor response of 18 mmHg, but did not in¯uence the release of 5-HT in the LC (Fig. 3). It is well established that low doses of KCN activate peripheral chemoreceptors and elicit hyperpnea, as well as cardiovascular changes in conscious rats [4,6,7,21]. We now found that chemoreceptor activation by a bolus injection of KCN enhances the release of serotonin in the LC. Furthermore, KCN evoked a respiratory re¯ex response and induced a pronounced transient rise in arterial BP. A rise in BP appears to be the typical response to cyanide-induced chemoreceptor activation in conscious rats [4,6,7,21], while decreases in BP can be found in anaesthetized rats [7,10,14]. The rise in BP, as well as the enhanced 5-HT release in response to KCN, were abolished in rats with transected chemoafferents, demonstrating that these responses were elicited by impulses reaching the CNS via sinoaortic afferents and not by a central action of cyanide. In this connection it is noteworthy that LC neurons also possess intrinsic chemo-receptive properties (for review see [13]). Since we have previously shown that pressor responses of approximately 40±60 mmHg enhance the release of 5-HT in the LC [16], it might be argued that the enhanced 5-HT release was due to the pressor response elicited by chemoreceptor stimulation. However, prolonged activation of chemoreceptors by slow infusion of KCN produced only a slight pressor response (5 mmHg), accompanied by
N. Singewald et al. / Neuroscience Letters 289 (2000) 17±20
Fig. 2. Effects of intravenous KCN infusion (15 mg/min) on 5-HT release in the LC and arterial blood pressure (BP). Mean release rate and mean BP in the three samples preceding infusion with KCN were taken as 100%. Bar denotes KCN infusion. SAD chemoreceptor denervation. Mean values ^ SEM *P , 0:05.
enhanced respiration rate throughout the infusion, but 5-HT release in the LC was even more increased by the prolonged chemoreceptor activation than by the bolus KCN injection. Furthermore, when a slight rise in BP (18 mmHg) was elicited by intravenous infusion of noradrenaline, 5-HT release in the LC was not in¯uenced. Hence, the enhanced extracellular concentration of 5-HT in the LC evoked by KCN was indeed due to ascending chemoreceptor information and not to the secondary pressor response. By increasing the dose of KCN from 15 to 30 mg/min, enhanced 5-HT release in the LC was prolonged and accompanied by a fall in BP. The latter effect was not solely related to activation of peripheral chemoreceptors, since the hypotension was similar in sham operated and SAD rats. The 5-HT response was abolished in SAD rats, indicating that also with this higher dose of KCN, mainly peripheral chemoreceptor activation contributed to the increase in LC 5-HT release. The fall in BP elicited by the high dose of KCN might be due to KCN toxicity leading to transient hypoxia and/or to cardioinhibitory effects. Hypoxia for example is known to produce nitric oxide-mediated vasodilatation [19]. The results of this study show that the 5-HT input to the LC is not only modulated by BP changes and stress (for review see [15]), but is also responsive to activation of
19
peripheral chemoreceptors. This ®nding suggests that alterations in the extracellular concentration of 5-HT may contribute to the modulation of the activity of LC neurons in response to chemosensory stimuli [2,5]. At present it is not clear how the chemoreceptor signals reach the LC. Chemoreceptor afferent ®bers are known to synapse in the nucleus tractus solitarii (NTS; commissural subnucleus and/or intermediolateral subnucleus), a principle site of the chemore¯ex pathway (for review see [21]). Indeed, cyanide-induced chemoreceptor stimulation enhances the discharge of NTS neurons [12]. Several neuronal pathways connected with the NTS are then thought to be modulated to produce appropriate responses to changes in chemoreceptor activation. There is a direct projection from the NTS to the LC [20], but also additional brain areas of the chemore¯ex neurocircuitry, such as the ventrolateral medulla, provide direct LC inputs and may be involved in conveying chemosensory information to the LC [5]. There is evidence that the LC may be involved in a chemore¯ex feedback loop modulating chemosensory input at the NTS in response to chemoreceptor activation [12]. Regarding a possible behavioral role of chemosensory stimuli, the well-documented change in the ®ring rate of LC neurons by chemoreceptor activation has been suggested to contribute to the associated arousal [4,5] and to serve as an
Fig. 3. Effects of intravenous infusion of KCN (30 mg/min) or noradrenaline (0.8±1 mg/kg per min) on 5-HT release in the LC and arterial blood pressure (BP). Mean release rate and mean BP in the three samples preceding infusion with drugs were taken as 100%. Bar denotes infusion of drugs. SAD chemoreceptor denervation. Mean values ^ SEM *P , 0:05, **P , 0:01.
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alarm reaction [2]. These ideas are particularly interesting, since the LC has been suggested to play a facilitatory role in the panic reaction [1]. Moreover, panic disorder patients are thought to react hyperresponsive to chemosensory stimuli and are particularly susceptible to the effects of hyperventilation (for review see [18]). It remains to be shown whether a possible dysfunctional LC neurotransmitter regulation in response to chemosensory stimuli is involved in such pathophysiological conditions. Taken together, we report that peripheral chemoreceptor activation enhances 5-HT release in the LC, indicating that 5-HT might be involved in the modulation of LC activity by ascending chemosensory information. These results extend previous observations that extracellular 5-HT in the LC responds to stimuli including baroreceptor signals and various somatosensory stimuli (for review see [15]), suggesting that this neurotransmitter integrates a large number of external and internal stimuli at the level of the LC to regulate the responsiveness of LC neurons in challenging situations. [1] Coplan, J.D. and Lydiard, R.B., Brain circuits in panic disorder, Biol. Psychiatry, 44 (1998) 1264±1276. [2] Elam, M., Yao, T., Thoren, P. and Svensson, T.H., Hypercapnia and hypoxia: chemoreceptor -mediated control of locus coeruleus neurons and splanchnic, sympathetic nerves, Brain Res., 222 (1981) 373±381. [3] Erickson, J.T. and Millhorn, D.E., Hypoxia and electrical stimulation of the carotid sinus nerve induce Fos-like immunoreactivity within catecholaminergic and serotoninergic neurons of the rat brainstem, J. Comp. Neurol., 348 (1994) 161±182. [4] Franchini, K.G. and Krieger, E.M., Cardiovascular responses of conscious rats to carotid body chemoreceptor stimulation by intravenous KCN, J. Autonom. Nerv. Syst., 42 (1993) 63±70. [5] Guyenet, P.G., Koshiya, N., Huangfu, D., Verberne, A.J.M. and Riley, T.A., Central respiratory control of A5 and A6 pontine noradrenergic neurons, Am. J. Physiol., 264 (1993) R1035±R1044. [6] Haibara, A.S., Colombari, E., Chianca, D.A., Bonagamba, L.G.H. and Machado, B.H., NMDA receptors in NTS are involved in bradycardic but not pressor response of chemore¯ex, Am. J. Physiol., 269 (1995) H1421±H1427 in press. [7] Hayward, L.F., Johnson, A.K. and Felder, R.B., Arterial chemore¯ex in conscious normotensive and hypertensive adult rats, Am. J. Physiol., 276 (1999) H1215±H1222.
[8] Hirooka, Y., Polson, J.W., Potts, P.D. and Dampney, R.A., Hypoxia-induced Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla, Neuroscience, 80 (1997) 1209±1224. [9] Kaehler, S.T., Singewald, N. and Philippu, A., The release of catecholamines in hypothalamus and locus coeruleus is modulated by peripheral chemoreceptors, NaunynSchmiedeberg's Arch. Pharmacol., 360 (1999) 428±434. [10] Kobayashi, M., Cheng, Z.B., Tanaka, K. and Nosaka, S., Is the aortic depressor nerve involved in arterial chemore¯exes in rats? J. Autonom. Nerv. Syst., 78 (1999) 38±48. [11] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1998. [12] Perez, H., Ruiz, S., Laurido, C. and Hernandez, A., Locus coeruleus-mediated inhibition of chemosensory responses in the rat nucleus tractus solitarius is mediated by a2-adrenoreceptors, Neurosci. Lett., 249 (1998) 37±40. [13] Pineda, J. and Aghajanian, G.K., Carbon dioxide regulates the tonic activity of locus coeruleus neurons by modulating a proton- and polyamine-sensitive inward recti®er potassium current, Neuroscience, 77 (1997) 723±743. [14] Sevoz, C., Callera, J.C., Machado, B.H., Hamon, M. and Laguzzi, R., Role of serotonin3 receptors in the nucleus tractus solitarii on the carotid chemore¯ex, Am. J. Physiol., 272 (1997) H1250±H1259. [15] Singewald, N. and Philippu, A., Release of neurotransmitters in the locus coeruleus, Prog. Neurobiol., 56 (1998) 237± 267. [16] Singewald, N., Kaehler, S.T., Hemeida, R. and Philippu, A., Release of serotonin in the rat locus coeruleus: effects of cardiovascular, stressful and noxious stimuli, Eur. J. Neurosci., 9 (1997) 556±562. [17] Singewald, N., Kaehler, S.T., Hemeida, R. and Philippu, A., In¯uence of excitatory amino acids on basal and sensory stimuli-induced release of 5-HT in the locus coeruleus, Br. J. Pharmacol., 123 (1998) 746±752. [18] Smoller, J.W., Pollack, M.H., Otto, M.W. and Rosenbaum, J.F., Panic anxiety, dyspnea, and respiratory disease, Am. J. Respir. Crit. Care. Med., 154 (1996) 6±17. [19] Sun, M.K. and Reis, D.J., Evidence that nitric oxide mediates the vasodepressor response to hypoxia in sinodenervated rats, Life Sci., 50 (1992) 555±565. [20] Van Bockstaele, E.J., Peoples, J. and Telegan, P., Efferent projections of the nucleus of the solitary tract to peri-locus coeruleus dendrites in rat brain: evidence for a monosynaptic pathway, J. Comp. Neurol., 412 (1999) 410±428. [21] Vasquez, E.C., Meyrelles, S.S., Mauad, H. and Cabral, A.M., Neural re¯ex regulation of arterial pressure in pathophysiological conditions: interplay among the barore¯ex, the cardiopulmonary re¯exes and the chemore¯ex, Braz. J. Med. Biol. Res., 30 (1997) 521±532.
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Peripheral chemoreceptor activation enhances 5-hydroxytryptamine release in the locus coeruleus of conscious rats Nicolas Singewald a,*, Dimitrios Kouvelas b, Stefan T. Kaehler a, Catrin Sinner a, Athineos Philippu a a
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, Peter-Mayr-Straûe 1, A-6020 Innsbruck, Austria b Department of Pharmacology, Aristotle University of Thessaloniki, Greece Received 10 April 2000; received in revised form 31 May 2000; accepted 31 May 2000
Abstract Intravenous bolus injection of KCN (40 mg) elicited brief but pronounced tachypnea, bradycardia and pressor response, and led to a 37% increase in 5-hydroxytryptamine (serotonin) (5-HT) release in the locus coeruleus (LC) of freely moving rats. Slow infusion of KCN (15 mg/min) for 10 min induced only a slight pressor response, but increased the respiration rate (139 breaths/min), as well as 5-HT release in the LC (160%) throughout the infusion. In rats with transected chemoreceptor afferents, neither injection or infusion of KCN changed 5-HT release, suggesting that in intact animals, the effect on extracellular 5-HT was due to activation of peripheral chemoreceptors. In summary, we report that peripheral chemoreceptor activation enhances 5-HT release in the LC, indicating that 5-HT might be involved in the modulation of LC activity by ascending chemosensory information. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 5-Hydroxytryptamine; Push-pull superfusion; Sinoaortic denervation; Chemore¯ex; Respiration; Blood pressure; Cyanide
Chemoreceptor activation by brief periods of hypoxia and hypercapnia have been shown to enhance the discharge rate of locus coeruleus (LC) noradrenergic neurons in anaesthetized animals [2,5]. Peripheral chemoreceptor stimulation by potassium cyanide (KCN) or CO2-saturated NaHCO3 enhances the release of noradrenaline in the LC of anaesthetized cats [9]. Both, enhanced ®ring of LC neurons as well as enhanced noradrenaline release in response to brief hypoxia or KCN can be reversed by transection of chemoreceptor afferents [2,5,9], showing that the activation of the LC noradrenergic system by these stimuli is indeed produced by peripheral chemoreceptor stimulation. In conscious animals, hypoxia has been shown to enhance cfos expression in the LC [3,8]. Although these and other studies show that the LC noradrenergic system is responsive to chemosensory stimuli, the afferent pathways and neurotransmitters involved in this modulation of LC neurons are little investigated. * Corresponding author. Tel.: 143-512-507-5601; fax: 143-512507-2931. E-mail address: [email protected] (N. Singewald).
Using the push-pull superfusion technique, we investigated in the present study whether 5-HT release in the LC of freely moving rats is in¯uenced by chemoreceptor activation. For this purpose, KCN was applied intravenously as a model of peripheral chemoreceptor activation [4,6,7,21] and its effects on blood pressure (BP), respiration and neurotransmitter release in the LC were studied. We have focused our interest on 5-HT release, since the LC is densely innervated by serotonergic ®bers (for review see [15]) and 5-HT has been suggested to play a role in the control of respiration in other parts of the brain, in particular the medulla (for review see [3]). Male Sprague±Dawley rats (280±300 g) were anaesthetized with diethylether and ketamine 50 mg/kg (i.p.). A guide cannula was stereotaxically inserted with its tip 2 mm above the LC [16]. The iliac artery and jugular vein were catheterized for recording of arterial BP, heart rate and intravenous infusions of drugs, respectively. In one group of rats peripheral chemoreceptor denervation involving transection of carotid sinus and aortic depressor nerves (SAD) [4]
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 24 1- 6
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N. Singewald et al. / Neuroscience Letters 289 (2000) 17±20
was carried out. Sham operated rats were subjected to the same surgical procedure without transection of nerves. At least ®ve days after surgery, the stylet of the guide cannula was replaced by a push-pull cannula which was 2 mm longer than the guide cannula thus reaching the LC. The coordinates were (mm): Anterior-posterior (AP) 0.8 posterior to interaural line, L 1.3, dorsal-ventral (DV) 2.8 above the interaural zero plane [11]. The LC was superfused with arti®cial cerebrospinal ¯uid (aCSF) at a rate of 14 ml/min. Superfusate was continuously collected in time periods of 10 min. 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) was determined in the superfusate by high pressure liquid chromatography (HPLC) with electrochemical detection [16]. Localization of the push-pull cannula was veri®ed histologically. Data are expressed as percentage of controls values. The mean release rate of 5-HT and mean arterial BP in the three samples preceding KCN administration were taken as control values. Statistical analysis was carried out by Friedman's analysis of variance followed by Wilcoxon's signed rank test for paired data. The mean basal release of 5-HT in sham operated rats was 1.4 ^ 0.1 fmol/min (mean ^ SEM, n 19) which is in good accordance with 5-HT release rates in the LC of intact rats that have not received sham operation [16,17]. SAD rats displayed similar release rates (1.5 ^ 0.2 fmol/min, mean ^
Fig. 1. Effects of intravenous KCN bolus injection (40 mg) on 5HT release in the LC and arterial blood pressure (BP). Mean release rate and mean BP in the three samples preceding KCN injection were taken as 100%. Arrow indicates KCN bolus injection. SAD chemoreceptor denervation. Mean ^ SEM *P , 0:05.
SEM, n 10), indicating that transection of chemoreceptor afferents had no effect on basal 5-HT release in the LC. Also resting BP did not differ in sham-operated (116 ^ 5 mmHg, mean ^ SEM, n 19) and SAD rats (111 ^ 6 mmHg, mean ^ SEM, n 10). In sham operated rats, intravenous bolus injection of KCN (40 mg) elicited a pressor response (160 mmHg, 152% from basal), which peaked within 10 s and lasted for 20 s (Fig. 1). KCN injection also led to brief bradycardia (2126 ^ 12 beats/min) and enhanced the respiration rate (146 ^ 5 breaths/min) for 2 min (not shown). KCN bolus injection enhanced the release rate of 5-HT in the LC by 37% (Fig. 1), while 5-HIAA out¯ow was not in¯uenced (not shown). In SAD rats, the effects of KCN on 5-HT release and arterial BP were abolished (Fig. 1). The slow intravenous infusion of KCN (15 mg/min) for 10 min elicited only a slight pressor response in sham operated rats which lasted for 2.5 min (Fig. 2). Respiration rate was increased throughout the infusion with KCN by 39 ^ 5 breaths/min (mean change ^ SEM, not shown). 5-HT release was enhanced by 60% during KCN infusion and was immediately normalized after termination of the infusion (Fig. 2). Enhanced 5-HT release as well as the pressor response to KCN infusion were abolished in SAD rats. Infusion of a higher dose of KCN (30 mg/min) led to a prolonged (20 min) increase in 5-HT release and decreased arterial BP by 10±15 mmHg (Fig. 3). The hypotensive response to the high dose of KCN was similar in sham operated and SAD rats, while the increase in 5-HT was abolished in SAD rats. Intravenous infusion of noradrenaline (0.8±1 mg/kg per min, n 6) led to a pressor response of 18 mmHg, but did not in¯uence the release of 5-HT in the LC (Fig. 3). It is well established that low doses of KCN activate peripheral chemoreceptors and elicit hyperpnea, as well as cardiovascular changes in conscious rats [4,6,7,21]. We now found that chemoreceptor activation by a bolus injection of KCN enhances the release of serotonin in the LC. Furthermore, KCN evoked a respiratory re¯ex response and induced a pronounced transient rise in arterial BP. A rise in BP appears to be the typical response to cyanide-induced chemoreceptor activation in conscious rats [4,6,7,21], while decreases in BP can be found in anaesthetized rats [7,10,14]. The rise in BP, as well as the enhanced 5-HT release in response to KCN, were abolished in rats with transected chemoafferents, demonstrating that these responses were elicited by impulses reaching the CNS via sinoaortic afferents and not by a central action of cyanide. In this connection it is noteworthy that LC neurons also possess intrinsic chemo-receptive properties (for review see [13]). Since we have previously shown that pressor responses of approximately 40±60 mmHg enhance the release of 5-HT in the LC [16], it might be argued that the enhanced 5-HT release was due to the pressor response elicited by chemoreceptor stimulation. However, prolonged activation of chemoreceptors by slow infusion of KCN produced only a slight pressor response (5 mmHg), accompanied by
N. Singewald et al. / Neuroscience Letters 289 (2000) 17±20
Fig. 2. Effects of intravenous KCN infusion (15 mg/min) on 5-HT release in the LC and arterial blood pressure (BP). Mean release rate and mean BP in the three samples preceding infusion with KCN were taken as 100%. Bar denotes KCN infusion. SAD chemoreceptor denervation. Mean values ^ SEM *P , 0:05.
enhanced respiration rate throughout the infusion, but 5-HT release in the LC was even more increased by the prolonged chemoreceptor activation than by the bolus KCN injection. Furthermore, when a slight rise in BP (18 mmHg) was elicited by intravenous infusion of noradrenaline, 5-HT release in the LC was not in¯uenced. Hence, the enhanced extracellular concentration of 5-HT in the LC evoked by KCN was indeed due to ascending chemoreceptor information and not to the secondary pressor response. By increasing the dose of KCN from 15 to 30 mg/min, enhanced 5-HT release in the LC was prolonged and accompanied by a fall in BP. The latter effect was not solely related to activation of peripheral chemoreceptors, since the hypotension was similar in sham operated and SAD rats. The 5-HT response was abolished in SAD rats, indicating that also with this higher dose of KCN, mainly peripheral chemoreceptor activation contributed to the increase in LC 5-HT release. The fall in BP elicited by the high dose of KCN might be due to KCN toxicity leading to transient hypoxia and/or to cardioinhibitory effects. Hypoxia for example is known to produce nitric oxide-mediated vasodilatation [19]. The results of this study show that the 5-HT input to the LC is not only modulated by BP changes and stress (for review see [15]), but is also responsive to activation of
19
peripheral chemoreceptors. This ®nding suggests that alterations in the extracellular concentration of 5-HT may contribute to the modulation of the activity of LC neurons in response to chemosensory stimuli [2,5]. At present it is not clear how the chemoreceptor signals reach the LC. Chemoreceptor afferent ®bers are known to synapse in the nucleus tractus solitarii (NTS; commissural subnucleus and/or intermediolateral subnucleus), a principle site of the chemore¯ex pathway (for review see [21]). Indeed, cyanide-induced chemoreceptor stimulation enhances the discharge of NTS neurons [12]. Several neuronal pathways connected with the NTS are then thought to be modulated to produce appropriate responses to changes in chemoreceptor activation. There is a direct projection from the NTS to the LC [20], but also additional brain areas of the chemore¯ex neurocircuitry, such as the ventrolateral medulla, provide direct LC inputs and may be involved in conveying chemosensory information to the LC [5]. There is evidence that the LC may be involved in a chemore¯ex feedback loop modulating chemosensory input at the NTS in response to chemoreceptor activation [12]. Regarding a possible behavioral role of chemosensory stimuli, the well-documented change in the ®ring rate of LC neurons by chemoreceptor activation has been suggested to contribute to the associated arousal [4,5] and to serve as an
Fig. 3. Effects of intravenous infusion of KCN (30 mg/min) or noradrenaline (0.8±1 mg/kg per min) on 5-HT release in the LC and arterial blood pressure (BP). Mean release rate and mean BP in the three samples preceding infusion with drugs were taken as 100%. Bar denotes infusion of drugs. SAD chemoreceptor denervation. Mean values ^ SEM *P , 0:05, **P , 0:01.
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N. Singewald et al. / Neuroscience Letters 289 (2000) 17±20
alarm reaction [2]. These ideas are particularly interesting, since the LC has been suggested to play a facilitatory role in the panic reaction [1]. Moreover, panic disorder patients are thought to react hyperresponsive to chemosensory stimuli and are particularly susceptible to the effects of hyperventilation (for review see [18]). It remains to be shown whether a possible dysfunctional LC neurotransmitter regulation in response to chemosensory stimuli is involved in such pathophysiological conditions. Taken together, we report that peripheral chemoreceptor activation enhances 5-HT release in the LC, indicating that 5-HT might be involved in the modulation of LC activity by ascending chemosensory information. These results extend previous observations that extracellular 5-HT in the LC responds to stimuli including baroreceptor signals and various somatosensory stimuli (for review see [15]), suggesting that this neurotransmitter integrates a large number of external and internal stimuli at the level of the LC to regulate the responsiveness of LC neurons in challenging situations. [1] Coplan, J.D. and Lydiard, R.B., Brain circuits in panic disorder, Biol. Psychiatry, 44 (1998) 1264±1276. [2] Elam, M., Yao, T., Thoren, P. and Svensson, T.H., Hypercapnia and hypoxia: chemoreceptor -mediated control of locus coeruleus neurons and splanchnic, sympathetic nerves, Brain Res., 222 (1981) 373±381. [3] Erickson, J.T. and Millhorn, D.E., Hypoxia and electrical stimulation of the carotid sinus nerve induce Fos-like immunoreactivity within catecholaminergic and serotoninergic neurons of the rat brainstem, J. Comp. Neurol., 348 (1994) 161±182. [4] Franchini, K.G. and Krieger, E.M., Cardiovascular responses of conscious rats to carotid body chemoreceptor stimulation by intravenous KCN, J. Autonom. Nerv. Syst., 42 (1993) 63±70. [5] Guyenet, P.G., Koshiya, N., Huangfu, D., Verberne, A.J.M. and Riley, T.A., Central respiratory control of A5 and A6 pontine noradrenergic neurons, Am. J. Physiol., 264 (1993) R1035±R1044. [6] Haibara, A.S., Colombari, E., Chianca, D.A., Bonagamba, L.G.H. and Machado, B.H., NMDA receptors in NTS are involved in bradycardic but not pressor response of chemore¯ex, Am. J. Physiol., 269 (1995) H1421±H1427 in press. [7] Hayward, L.F., Johnson, A.K. and Felder, R.B., Arterial chemore¯ex in conscious normotensive and hypertensive adult rats, Am. J. Physiol., 276 (1999) H1215±H1222.
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