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Brainstem mechanisms controlling cardiovascular reflexes in channel catfish
Comparative Biochemistry and Physiology, Part A 170 (2014) 1–5
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Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Brainstem mechanisms controlling cardiovascular reflexes in channel catfish J. Turesson b, M.S. Hedrick a, L. Sundin c, M.L. Burleson a,⁎ a b c
Department of Biological Sciences, University of North Texas, P.O. Box 305220, Denton, TX, 76203–5220, USA Department of Pediatrics, Karolinska Institute, Stockholm, Sweden Department of Zoophysiology, Göteborg University, P.O. Box 463, S-405 30, Gothenburg, Sweden
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Article history: Received 19 August 2013 Received in revised form 24 December 2013 Accepted 6 January 2014 Available online 13 January 2014 Keywords: Chemosensitivity, Central heart rate, blood pressure, general visceral nucleus, fish, hypoxia, kainic acid, kynurenic acid, kainic acid Receptors, NMDA, AMPA
a b s t r a c t Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Specific regions of the brain have been shown to control cardiovascular variables in vertebrates and much of the current research on fishes focuses on functional anatomy of these regions (Le Mével et al., 2012). Sensory information from various peripheral receptors is routed through cardioventilatory control centers where it is integrated and used to modify motor output to effectors. O2-sensitive chemoreceptors are one kind of important sensory receptors that send their information to cardiovascular and ventilatory control centers in the medulla that, in turn, alter cardioventilatory performance patterns and allow animals to respond to changes in O2 availability and demand. Most of what is known about central control of cardiovascular function comes from mammalian studies and very little comparative data are available. The regulation of a double circulatory circuit (systemic and pulmonary) under different pressures in mammals likely involves much more complex central neural circuitry than the single circuit circulatory system of water breathing fishes. Since the cardioventilatory control networks of air-breathing vertebrates evolved from the more primitive water-breathers, fish should provide a good model system to study the central mechanisms of cardioventilatory control. Information regarding the locations of central integration centers and the neurochemicals involved, in a variety of different species of vertebrates, will lead to a better understanding of the evolution of cardiovascular control mechanisms, effects of environmental variables on central mechanisms ⁎ Corresponding author. Tel.: +1 940 369 5142. E-mail address: [email protected] (M.L. Burleson). 1095-6433/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2014.01.003
(i.e. chronic hypoxia) and possible treatment strategies for human disorders. Most of the peripheral receptors (mechano- and chemo-) controlling cardiovascular function in fishes appear to be located in the gill arches and innervated by cranial nerves IX and X (Burleson et al., 1992 for review). Neuroanatomical studies have shown that the sensory axons in these nerves terminate in an area described variously as the general visceral nucleus (Kanwal and Caprio, 1987) or vagal sensory area (Sundin et al., 2003a,b) which is homologous to the nucleus tractus solitarius, the major cardioventilatory integration center in mammals (Le Mével et al., 2012). Previous studies have shown this region in fishes to be involved in the modulation of the cardioventilatory responses to hypoxia (Sundin et al., 2003a,b; Turesson and Sundin, 2003; Turesson et al., 2010). Glutamate appears to be the most important neurotransmitter for integration of sensory input and motor output from the central cardioventilatory control regions in mammals (Ohtake et al., 1998; Lin et al., 1999) as well as ectothermic vertebrates such as amphibians (Hoffman et al., 1993). Glutamate-containing cells have been identified using immunohistochemical methods in the general visceral nucleus of catfish and sculpin and microinjections of glutamate have been shown to alter cardioventilatory variables (Sundin et al., 2003b; Turesson and Sundin, 2003; Turesson et al., 2010). Glutamatergic neurons show an extensive distribution through all of the areas of the brainstem of lamprey (Petromyzon marinus) in all of the regions/ structures involved with cardioventilatory control (Villar-Cerviño et al., 2013). Coordination of ventilation and perfusion is especially critical in water breathing vertebrates where the ventilation to perfusion
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ratio may be 10 or greater and often quite variable (Taylor et al., 1999). Thus, central mechanisms must play an important role in coordinating the cardiovascular and ventilatory systems. In a previous paper (Sundin et al., 2003a) we focused on the ventilatory effects of kainic and kynurenic acid microinjections into the brainstem of anesthetized channel catfish. The purpose of this paper was to further analyze those experiments. This paper analyzes the cardiovascular effects of microinjections of kainic and kynurenic acid to identify central areas and mechanisms controlling normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia in channel catfish. 2. Material and methods 2.1. Experimental animals Channel catfish, Ictalurus punctatus, (~ 400–700 g) were obtained from a local commercial supplier. They were transported to the laboratory and maintained in 1900 l fiberglass tanks filled with recirculating, dechlorinated water and equipped with biological filters. Fish were maintained at 22–24 °C and a 12L/12D cycle. They were fed commercial catfish chow ad libitum every 2–3 days and were allowed 1 week to acclimate to laboratory conditions prior to experimentation. UTA IACUC-approved protocol #01-6. 2.2. Surgery Fish (N = 14) were anesthetized with MS 222 (1 g/10 l dechlorinated water). A cannula (PE 50) was implanted in the afferent branchial artery of the third gill arch (Axelsson and Fritsche, 1994) to monitor ventral aorta blood pressure (PVA) and heart rate (fH) with a pressure transducer. The brainstem was exposed through an opening made in the skull (approximately 1 × 1 cm) with a small, high-speed circular saw. After surgery, the fish were secured in a stereotaxic device in a plexiglass chamber with ventilation assisted as previously described (Burleson and Smatresk, 2000). The level of anesthesia was reduced to 0.2 g/10 l water and cardioventilatory variables were monitored until ventilation and blood pressure were stable for at least 30 min before beginning the protocol. 2.3. Procedures Blood pressure traces were recorded using computers equipped with analog–digital converters and associated software (LabView and Windaq) for data acquisition and post-acquisition analyses. Aquatic hypoxia was induced by bubbling the water with N2. Oxygen tension was monitored using a submersible O2 electrode and associated meter (YSI). Micropipettes with tip diameters approximately 10–20 μm were constructed from glass capillary tubing (A-M Systems, o.d. 1.2 mm, i.d. 0.9 mm) using a pipette puller (Sutter Instruments). A piezo
micromanipulator (PM 10, World Precision Instruments) was used to position the micropipettes. Bilateral microinjections into the primary general visceral nucleus (nGV) of the medulla (Fig. 1) were made using a picospritzer (General Valve). Using the obex as a reference point, injections were made at sites 0.3 mm to 1.0 mm lateral and rostral and up to 1.9 mm depth. Injection volume was measured by monitoring the fluid meniscus in the micropipette with a microscope equipped with an ocular reticule. Microinjection volume was calculated by applying the formula for volume of a cylinder (volume = π × radius2 × height) to the internal dimensions of the micropipette. Volumes of microinjections ranged from 36 to 108 nl. Drugs used were: kainic acid (0.01 mM), for chemical lesion of neurons and kynurenic acid (10 mM), a nonselective NMDA/AMPA/kainate glutamate antagonist. Drugs were dissolved in mock CSF with the following composition in mm/l: 120 NaCl, 4 KCl, 1 MgSO4, 1 CaCl2 and 10 NaHCO3 adjusted to pH 7.4. Control injections of equivalent volumes were made with mock CSF. Fluorescent microspheres (diameter = 0.05 μm; Polysciences) were used to verify injection sites (Sundin et al., 2003a). 2.4. Protocol Saline control microinjections were done first. Cardiovascular variables were recorded for several minutes during normoxia then the fish was exposed to rapid, acute hypoxia (PO2 ~ 30 Torr) for approximately 3 min before returning to normoxia. Following control microinjections with mock CSF, microinjections of either kainic acid or kynurenic acid were made and the fish exposed to normoxia and hypoxia as before. At the end of an experiment fish were killed with an overdose of anesthesia (MS-222). Heparin (0.2 ml, 5000 IU) was injected by cardiac puncture then fish were exsanguinated via the ventral aorta with approximately 60 ml of heparinized saline. Following exsanguinations, fish were perfused with 20 ml of 4% formaldehyde in phosphate buffered saline. Brains were removed and stored in 4% formaldehyde solution for anatomical studies. Serial transverse sections (20–40 μm) were cut and air-dried overnight on gelatine-coated glass slides. Sections were cover-slipped and viewed with a fluorescent microscope to confirm injection sites and estimate spread of injected material (see Sundin et al., 2003a). The effects of hypoxia and microinjections on heart rate and blood pressure were analyzed with a 2-way ANOVA. Specific effects (i.e. the effects of hypoxia after an injection) were analyzed using the least significant difference test for planned post hoc comparisons (P b 0.05). 3. Results 3.1. Mock CSF controls and response to hypoxia Control microinjections of mock CSF did not alter the cardiovascular effects of hypoxia (Figs. 2, 3). Fish responded as expected to hypoxia with a significant bradycardia after microinjections of mock CSF. The optic tectum lateral line lobe facial lobe vagal lobe obex
cerebellum
spinal cord
4th ventricle
injection sites
Fig. 1. Drawing of channel catfish brain (dorsal view) with major structures labeled and showing the approximate locations of microinjections (black dots).
J. Turesson et al. / Comparative Biochemistry and Physiology, Part A 170 (2014) 1–5
normoxia hypoxia
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Fig. 2. Mean cardiovascular responses to hypoxia after control (mock CSF) and kainic acid microinjections. Values are means ± S.E.M. (N = 6). * indicates statistically significant difference from normoxic control.
difference in blood pressure between normoxia and hypoxia was not statistically significant. 3.2. Kainic acid The cardiovascular effects of kainic acid microinjections are shown in Fig. 2 (N = 6). Heart rate in normoxia was 108 ± 6 beats min− 1 and decreased significantly to 82 ± 11 beats min −1 in aquatic hypoxia (P b 0.05). Following bilateral microinjection of kainic acid, heart rate decreased to 85 ± 2 beats min −1 (P b 0.05) in normoxia and did not change further during exposure to hypoxia (Fig. 2). Blood pressure was 33.3 ± 1.2 cm H2O in normoxia and was not affected by aquatic hypoxia, or by kainic acid and exposure to normoxia or hypoxia (Fig. 2). 3.3. Kynurenic acid The cardiovascular effects of kynurenic acid microinjections are shown in Fig. 3 (N = 8). Heart rate was 96 ± 5 beats min −1 in normoxia and hypoxia caused a significant bradycardia to 65 ± 6 beats min −1 (P b 0.05). After kynurenic acid injections normoxic heart rate was 76 ± 7 beats min −1 which was significantly lower than control heart rate. Following microinjection of kynurenic acid, exposure to aquatic hypoxia produced a significant bradycardia to 59 ± 9 beats min −1. Blood pressure was 24.4 ± 2.1 cm H2O in normoxia and was not affected by aquatic hypoxia, or by kynurenic acid and exposure to normoxia or hypoxia (Fig. 3).
3.4. Microscopy Microscopic examination of fixed brain slices confirmed that microinjections were made into the sensory region and not into underlying motor neurons. Spread of injections in the vertical plane was approximately 400 μm, independent of injection volume and between 650 and 1400 μm in the horizontal plane (Sundin et al., 2003a). 4. Discussion This study has shown that nGV microinjections of the ionotropic glutamate receptor antagonist kynurenic acid or chemical lesions of glutamatergic neurons with kainic acid significantly lower heart rate in normoxia. Furthermore, kainic acid, but not kynurenic acid, also blocks the bradycardia in response to aquatic hypoxia. These results suggest that central glutamatergic neurons exert a tonic excitatory control of HR in normoxia and glutamatergic receptors in the nGV play an integrative role in the cardiovascular response to aquatic hypoxia in the catfish. 4.1. Critique of methods First, in order to examine the role of centrally-microinjected neurochemicals on cardiovascular function in catfish, it was necessary to use lightly-anesthetized, spontaneously-breathing animals. There are some drawbacks to using MS 222 anesthesia, but anesthesia is necessary in
normoxia hypoxia 120
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100
* 80
*
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60 40 20
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30 25 20 15 10 5 0
0 control
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Fig. 3. Mean cardiovascular responses to hypoxia after control (mock CSF) and kynurenic acid microinjections. Values are means ± S.E.M. (N = 8). * indicates statistically significant difference from normoxic control. + indicates statistically significant difference from normoxic kynurenic acid.
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order to perform the microinjection experiments as we have done in this study. An alternative to this procedure would be to decerebrate the animals, thereby eliminating the need for anesthesia. However, previous studies have shown that decerebration causes unacceptable changes in blood pressure due to hemorrhage (Burleson and Smatresk, 1987; Sundin et al., 2003a,b). Resting heart rate in anesthetized, spontaneously-breathing catfish is considerably higher than either decerebrate or unanesthetized catfish, while anesthesia causes hypotension compared with unanesthetized fish. Anesthesia also attenuates the blood pressure and heart rate response to hypoxia (Burleson and Smatresk, 2000). The elevated heart rate under normoxic conditions is likely caused by the sympathetic cardiac stimulation by MS 222 (Smith, 1974) or by a direct excitatory effect on the central nervous system that occurs at low concentrations of MS 222 (Hedrick and Winmill, 2003). Another explanation for the high resting heart rate is that high water flow velocities associated with the assisted ventilation may have elevated resting heart rate via a mechanoreceptor reflex (Taylor and Butler, 1982; Barrett and Taylor, 1984). Despite these drawbacks, however, chemoreceptor stimulation by hypoxia produces cardioventilatory reflexes to aquatic hypoxia, albeit an attenuated response, compared with that of unanesthetized catfish (Burleson and Smatresk, 2000; Sundin et al., 2003a). Secondly, another critique is that both of the drugs used have effects on receptors and cells other than glutamatergic neurons and specific glutamate pathways. Kynurenic acid and its metabolites are now being shown to affect a number of different pathways. It stimulates the G-protein-coupled receptor GPR35 and, although controversial, may also inhibit α7 nicotinic acetylcholine receptors (Stone et al., 2013). Cell death by kainic acid releases a variety of excitatory amino acids, cytokines and inflammatory chemicals (Zhang and Zhu, 2011). All of these have potential to affect central control of cardiac function and could account for differences in the cardiovascular responses to kynurenic and kainic acid. Thirdly, despite negative immunohistochemical evidence for glutamate in the nGV, there were positive results for the NMDA receptor subunit 1 and both kynurenic and kainic acid had significant effects on cardiovascular variables. The lack of glutamate immunoreactivity contrasts with positive results in sculpin (Sundin et al., 2003b). It may be due to an error in protocol or processing of the tissues or variation between species but clearly needs to be re-examined. 4.2. Effects of kynurenic acid and kainic acid microinjections on heart rate and blood pressure Blocking glutamatergic transmission with both kainic and kynurenic acid resulted in a decrease in heart rate, suggesting that under normoxic conditions there is tonic excitatory glutamatergic input maintaining an elevated heart rate in lightly-anesthetized catfish. Loss of glutamatergic input by chemical lesions of glutamatergic neurons with kainic acid or blocking ionotropic glutamate receptors with kynurenate produced a similar degree of bradycardia under normoxic conditions. The results with glutamatergic antagonists were somewhat unexpected because microinjection of the agonist glutamate has been reported to result in bradycardia. For example, microinjection of glutamate into the nGV of shorthorn sculpin (Myoxocephalus scorpius) causes a reflex bradycardia (Sundin et al., 2003b). This reflex bradycardia is blocked by the NMDA receptor antagonist MK801 microinjected into the same glutamatesensitive site in the nGV suggesting that NMDA receptors mediate this response (Sundin et al., 2003b). In the present study, glutamate microinjections into the nGV of catfish produced no heart rate response (data not shown) and did not produce any ventilatory response in a previous study with catfish (Sundin et al., 2003a). The lack of cardioventilatory response is consistent with the finding that the nGV does not exhibit glutamate immunoreactivity in the catfish (Sundin et al., 2003a). Thus, the integration of cardioventilatory reflexes in the nGV appears to differ markedly between shorthorn sculpin and channel catfish.
A possible explanation for the results with kainic and kynurenic acid is that, in catfish, the nGV serves as an important region for integration of sensory input as well as integrating inputs from other regions of the brain. Because MS 222 anesthesia elevates heart rate, presumably through a sympathetic mechanism (Smith, 1974), the data in the present study indicate that glutamatergic blockade within the nGV probably reduces sympathetic outflow to the heart in catfish. Bradycardia induced by hypoxia or by glutamate microinjected into the nGV is primarily mediated by increased parasympathetic vagal input to the heart since it is blocked by atropine (Taylor et al., 1999; Sundin et al., 2003b). However, branchial nerve section in atropinized catfish fails to completely abolish the hypoxic bradycardia (Burleson and Smatresk, 1990) indicating that withdrawal of sympathetic tone to the heart may be involved in this reflex. Whether this would occur in unanesthetized catfish cannot be addressed by the present study. 4.3. Integration of cardiovascular reflexes in hypoxia Aquatic hypoxia normally evokes an increase in ventilation and a reflex bradycardia in fish owing to stimulation of chemoreceptors located on the gills (Nilsson, 1984; Burleson et al., 1992). Substantial evidence indicates that gill chemoreceptors are both externally and internally-oriented in order to respond to aquatic and blood oxygen status, respectively. Gill chemoreceptors are innervated by sensory branches of cranial nerves IX and X which terminate in the nGV to form the first-order relay for chemoreceptor-mediated reflexes. In teleosts, the nGV forms a continuous dorso-lateral column on either side of the 4th ventricle in the medulla, into which the sensory fibers from visceral structures terminate (Meek and Nieuwenhuys, 1998). Because the nGV represents the termination of gill chemoreceptor pathways in fish, central stimulation of this pathway by glutamate mimics the bradycardia and ventilatory increase evoked by peripheral chemoreceptor stimulation by hypoxia (Sundin et al., 2003a,b). In shorthorn sculpin, hypoxia or cyanide-evoked increases in ventilation are abolished by systemic injection of the NMDA receptor antagonist MK-801, but the bradycardia resulting from these stimuli is not blocked by MK-801 (Turesson and Sundin, 2003). This indicates that central glutamatergic pathways that modulate ventilatory and cardiovascular differ in response to peripheral chemoreceptor stimulation. In a previous study with catfish (Sundin et al., 2003a), microinjections of kainic acid into the nGV abolished breathing in normoxic conditions whereas kynurenic acid microinjections in the same area blocked the respiratory increase in hypoxia. In the present study, destruction of glutamatergic neurons in the nGV with kainic acid blocked the bradycardia in response to aquatic hypoxia, but kynurenic acid microinjections did not. A major difference between these two treatments is that while kainic acid destroys glutamatergic neurons and prevents the activation of both ionotropic and metabotropic glutamate receptors, kynurenic acid blocks only ionotropic receptors. This would suggest that metabotropic receptors may be involved in the integration of hypoxic reflexes within the nGV in catfish. Group I metabotropic glutamate receptors mediate cardiovascular reflexes within the NTS of urethane-anesthetized rats (Foley et al., 1999). Although control of heart rate by glutamatergic receptors within the nGV of catfish is indicated by the results of this study, the precise mechanisms and pathways of regulation are not clear. Our histological results (Sundin et al., 2003b) indicate that we were successful in blocking glutamatergic pathways within the sensory region of the medulla and did not evoke these responses by activation of underlying ventral motor areas. Because heart rate decreased in response to blockade of ionotropic receptors or loss of glutamatergic neurons within the nGV, this region represents a source or pathway for tonic glutamate release that maintains an elevated heart rate. This is consistent with the fact that glutamate microinjections in this region did not have any effect on heart rate in catfish (data not shown), whereas microinjections of glutamate in the nGV of shorthorn sculpin produced a bradycardia (Sundin
J. Turesson et al. / Comparative Biochemistry and Physiology, Part A 170 (2014) 1–5
et al., 2003b). As a comparison to the glutamate studies, intracerebroventricular injections of various neuropeptides in unanesthetized rainbow trout either had no effect or increased heart rate (Le Mével et al., 2012). These neuropeptides have been immunohistochemically localized to the same central cardioventilatory control areas and also work through G-protein-coupled receptors. It is not clear if the tonic glutamatergic input from the nGV occurs under the specific conditions of this study (i.e. light anesthesia), or if this is unique to catfish. Given the differences in cardioventilatory responses with microinjections of glutamatergic drugs into similar areas of the medulla between shorthorn sculpin (Sundin et al., 2003b: Turesson et al., 2010) and channel catfish (Sundin et al., 2003a, present study) under similar experimental conditions, it appears that there are substantial species differences in cardioventilatory regulation within the medulla. Acknowledgments This work was supported in part by grants from the National Institutes of Health (HL076205-01), the Swedish Research Council, Helge Axelson Johnson Stiftelse, Stiftelsen Längmanska Kulturfonden, and Wilhelm and Martina's Research Foundation. We thank Gunilla Rydgren for histological preparations of the brains and Adam Brooks for illustration of the catfish brain. References Axelsson, M., Fritsche, R., 1994. Cannulation techniques. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes, Analytical Techniques, vol. 3. Elsevier, Amsterdam, pp. 17–36. Barrett, D.J., Taylor, E., 1984. Changes in heart rate during progressive hypoxia in the dogfish Scyliorhinus canicula L.: evidence for a venous oxygen receptor. Comp. Biochem. Physiol. 78, 697–703. Burleson, M.L., Smatresk, N.J., 1987. The effect of decerebration and anesthesia on the reflex responses to hypoxia in catfish. Can. J. Zool. 67, 630–635. Burleson, M.L., Smatresk, N.J., 1990. Effects of sectioning cranial nerves IX and X on cardiovascular and ventilatory reflex responses to hypoxia and NaCN in channel catfish. J. Exp. Biol. 154, 407–420. Burleson, M.L., Smatresk, N.J., 2000. Branchial chemoreceptors mediate ventilatory responses to hypercapnic acidosis in channel catfish. Comp. Biochem. Physiol. A 125, 403–414. Burleson, M.L., Smatresk, N.J., Milsom, W.K., 1992. Afferent inputs associated with cardioventilatory control in fish. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish
5
Physiology. The Cardiovascular System, vol. XXIIB. Academic Press, New York, pp. 390–426. Foley, C.M., Vogl, H.W., Mueller, P.J., Hay, M., Hasser, E.M., 1999. Cardiovascular response to group I metabotropic glutamate receptor activation in NTS. Am. J. Physiol. 276, R1469–R1478. Hedrick, M.S., Winmill, R.E., 2003. Excitatory and inhibitory effects of tricaine (MS-222) on fictive breathing in isolated bullfrog brain stem. Am. J. Physiol. 284, R405–R412. Hoffman, A., Brazil Romero, S.M., de Oliveira, L.M., 1993. Agonistic behavior and its cardiovascular components elicited by microinjection of L-glutamic acid into the basal midbrain of the toad Bufo paracnemis. Brain Behav. Evol. 41, 316–325. Kanwal, J.S., Caprio, J., 1987. Central projections of the glossopharyngeal and vagal nerves in the channel catfish, Ictalurus punctatus: clues to differential processing of visceral inputs. J. Comp. Neurol. 264, 216–230. Le Mével, J.C., Lancien, F., Mimassi, N., Conlon, J.M., 2012. Brain neuropeptides in central ventilatory and cardiovascular regulation in trout. Front. Endocrinol. 3, 124. http:// dx.doi.org/10.3389/fendo.2012.00124. Lin, H.-C., Wan, F.-J., Tseng, C.-J., 1999. Modulation of cardiovascular effects produced by nitric oxide and ionotropic glutamate receptor interaction in the nucleus tractus solitarii of rats. Neuropharmacology 38, 935–941. Meek, J., Nieuwenhuys, R., 1998. Holosteans and teleosts. In: Nieuwenhuys, R., Ten Donkelaar, H.I., Nicholson, C. (Eds.), The Central Nervous System of Vertebrates, vol. 2. Springer, Berlin, pp. 759–937. Nilsson, S., 1984. Innervation and pharmacology of the gills. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. 10A. Academic Press, New York, pp. 185–227. Ohtake, P.J., Torres, J.E., Gozal, Y.M., Graff, G.R., Gozal, D., 1998. NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat. J. Appl. Physiol. 84, 853–861. Smith, D.G., 1974. Sympathetic cardiac stimulation in Bufo marinus under MS-222 anesthesia. Am. J. Physiol. 226, 367–370. Stone, T.W., Stoy, N., Darlington, G.L., 2013. An expanding range of targets for kynurenine metabolites of tryptophan. Trends Pharmacol. Sci. 34, 136–143. Sundin, L., Turesson, J., Burleson, M., 2003a. Identification of central mechanisms vital for breathing in the channel catfish, Ictalurus punctatus. Respir. Physiol. Neurobiol. 138, 77–86. Sundin, L., Turesson, J., Taylor, E.W., 2003b. Evidence for glutamatergic mechanisms in the vagal sensory pathway initiating cardiorespiratory reflexes in the shorthorn sculpin Myoxocephalus scorpius. J. Exp. Biol. 206, 867–876. Taylor, E.W., Butler, P.J., 1982. Nervous control of heart rate: activity in the cardiac vagus of the dogfish. J. Appl. Physiol. 43, 1330–1335. Taylor, E.W., Jordan, D., Coote, J.H., 1999. Central control of the cardiovascular and respiratory systems and their interactions in vertebrates. Physiol. Rev. 79, 855–916. Turesson, J., Sundin, L., 2003. N-methyl-D-aspartate receptors mediate chemoreflexes in the shorthorn sculpin Myoxocephalus scorpius. J. Exp. Biol. 206, 1251–1259. Turesson, J., Johansson, M., Sundin, L., 2010. Involvement of non-NMDA receptors in central mediation of chemoreflexes in the shorthorn sculpin, Myoxocephalus scorpius. Respir. Physiol. Neurobiol. 172 (3), 83–93. Villar-Cerviño, V., Barreiro-Iglesias, A., Fernández-lópez, B., Mazan, S., Rodicio, M.C., Anadón, R., 2013. Glutamatergic neuronal populations in the brainstem of the sea lamprey, Petromyzon marinus: an in situ hybridization and immunocytochemical study. J. Comp. Neurol. 521, 522–557. http://dx.doi.org/10.1002/cne.23189. Zhang, X.M., Zhu, J., 2011. Kainic acid-induced neurotoxicity: targeting glial responses and glia-derived cytokines. Curr. Neuropharmacol. 9, 388–398.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Brainstem mechanisms controlling cardiovascular reflexes in channel catfish J. Turesson b, M.S. Hedrick a, L. Sundin c, M.L. Burleson a,⁎ a b c
Department of Biological Sciences, University of North Texas, P.O. Box 305220, Denton, TX, 76203–5220, USA Department of Pediatrics, Karolinska Institute, Stockholm, Sweden Department of Zoophysiology, Göteborg University, P.O. Box 463, S-405 30, Gothenburg, Sweden
a r t i c l e
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Article history: Received 19 August 2013 Received in revised form 24 December 2013 Accepted 6 January 2014 Available online 13 January 2014 Keywords: Chemosensitivity, Central heart rate, blood pressure, general visceral nucleus, fish, hypoxia, kainic acid, kynurenic acid, kainic acid Receptors, NMDA, AMPA
a b s t r a c t Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Specific regions of the brain have been shown to control cardiovascular variables in vertebrates and much of the current research on fishes focuses on functional anatomy of these regions (Le Mével et al., 2012). Sensory information from various peripheral receptors is routed through cardioventilatory control centers where it is integrated and used to modify motor output to effectors. O2-sensitive chemoreceptors are one kind of important sensory receptors that send their information to cardiovascular and ventilatory control centers in the medulla that, in turn, alter cardioventilatory performance patterns and allow animals to respond to changes in O2 availability and demand. Most of what is known about central control of cardiovascular function comes from mammalian studies and very little comparative data are available. The regulation of a double circulatory circuit (systemic and pulmonary) under different pressures in mammals likely involves much more complex central neural circuitry than the single circuit circulatory system of water breathing fishes. Since the cardioventilatory control networks of air-breathing vertebrates evolved from the more primitive water-breathers, fish should provide a good model system to study the central mechanisms of cardioventilatory control. Information regarding the locations of central integration centers and the neurochemicals involved, in a variety of different species of vertebrates, will lead to a better understanding of the evolution of cardiovascular control mechanisms, effects of environmental variables on central mechanisms ⁎ Corresponding author. Tel.: +1 940 369 5142. E-mail address: [email protected] (M.L. Burleson). 1095-6433/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2014.01.003
(i.e. chronic hypoxia) and possible treatment strategies for human disorders. Most of the peripheral receptors (mechano- and chemo-) controlling cardiovascular function in fishes appear to be located in the gill arches and innervated by cranial nerves IX and X (Burleson et al., 1992 for review). Neuroanatomical studies have shown that the sensory axons in these nerves terminate in an area described variously as the general visceral nucleus (Kanwal and Caprio, 1987) or vagal sensory area (Sundin et al., 2003a,b) which is homologous to the nucleus tractus solitarius, the major cardioventilatory integration center in mammals (Le Mével et al., 2012). Previous studies have shown this region in fishes to be involved in the modulation of the cardioventilatory responses to hypoxia (Sundin et al., 2003a,b; Turesson and Sundin, 2003; Turesson et al., 2010). Glutamate appears to be the most important neurotransmitter for integration of sensory input and motor output from the central cardioventilatory control regions in mammals (Ohtake et al., 1998; Lin et al., 1999) as well as ectothermic vertebrates such as amphibians (Hoffman et al., 1993). Glutamate-containing cells have been identified using immunohistochemical methods in the general visceral nucleus of catfish and sculpin and microinjections of glutamate have been shown to alter cardioventilatory variables (Sundin et al., 2003b; Turesson and Sundin, 2003; Turesson et al., 2010). Glutamatergic neurons show an extensive distribution through all of the areas of the brainstem of lamprey (Petromyzon marinus) in all of the regions/ structures involved with cardioventilatory control (Villar-Cerviño et al., 2013). Coordination of ventilation and perfusion is especially critical in water breathing vertebrates where the ventilation to perfusion
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ratio may be 10 or greater and often quite variable (Taylor et al., 1999). Thus, central mechanisms must play an important role in coordinating the cardiovascular and ventilatory systems. In a previous paper (Sundin et al., 2003a) we focused on the ventilatory effects of kainic and kynurenic acid microinjections into the brainstem of anesthetized channel catfish. The purpose of this paper was to further analyze those experiments. This paper analyzes the cardiovascular effects of microinjections of kainic and kynurenic acid to identify central areas and mechanisms controlling normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia in channel catfish. 2. Material and methods 2.1. Experimental animals Channel catfish, Ictalurus punctatus, (~ 400–700 g) were obtained from a local commercial supplier. They were transported to the laboratory and maintained in 1900 l fiberglass tanks filled with recirculating, dechlorinated water and equipped with biological filters. Fish were maintained at 22–24 °C and a 12L/12D cycle. They were fed commercial catfish chow ad libitum every 2–3 days and were allowed 1 week to acclimate to laboratory conditions prior to experimentation. UTA IACUC-approved protocol #01-6. 2.2. Surgery Fish (N = 14) were anesthetized with MS 222 (1 g/10 l dechlorinated water). A cannula (PE 50) was implanted in the afferent branchial artery of the third gill arch (Axelsson and Fritsche, 1994) to monitor ventral aorta blood pressure (PVA) and heart rate (fH) with a pressure transducer. The brainstem was exposed through an opening made in the skull (approximately 1 × 1 cm) with a small, high-speed circular saw. After surgery, the fish were secured in a stereotaxic device in a plexiglass chamber with ventilation assisted as previously described (Burleson and Smatresk, 2000). The level of anesthesia was reduced to 0.2 g/10 l water and cardioventilatory variables were monitored until ventilation and blood pressure were stable for at least 30 min before beginning the protocol. 2.3. Procedures Blood pressure traces were recorded using computers equipped with analog–digital converters and associated software (LabView and Windaq) for data acquisition and post-acquisition analyses. Aquatic hypoxia was induced by bubbling the water with N2. Oxygen tension was monitored using a submersible O2 electrode and associated meter (YSI). Micropipettes with tip diameters approximately 10–20 μm were constructed from glass capillary tubing (A-M Systems, o.d. 1.2 mm, i.d. 0.9 mm) using a pipette puller (Sutter Instruments). A piezo
micromanipulator (PM 10, World Precision Instruments) was used to position the micropipettes. Bilateral microinjections into the primary general visceral nucleus (nGV) of the medulla (Fig. 1) were made using a picospritzer (General Valve). Using the obex as a reference point, injections were made at sites 0.3 mm to 1.0 mm lateral and rostral and up to 1.9 mm depth. Injection volume was measured by monitoring the fluid meniscus in the micropipette with a microscope equipped with an ocular reticule. Microinjection volume was calculated by applying the formula for volume of a cylinder (volume = π × radius2 × height) to the internal dimensions of the micropipette. Volumes of microinjections ranged from 36 to 108 nl. Drugs used were: kainic acid (0.01 mM), for chemical lesion of neurons and kynurenic acid (10 mM), a nonselective NMDA/AMPA/kainate glutamate antagonist. Drugs were dissolved in mock CSF with the following composition in mm/l: 120 NaCl, 4 KCl, 1 MgSO4, 1 CaCl2 and 10 NaHCO3 adjusted to pH 7.4. Control injections of equivalent volumes were made with mock CSF. Fluorescent microspheres (diameter = 0.05 μm; Polysciences) were used to verify injection sites (Sundin et al., 2003a). 2.4. Protocol Saline control microinjections were done first. Cardiovascular variables were recorded for several minutes during normoxia then the fish was exposed to rapid, acute hypoxia (PO2 ~ 30 Torr) for approximately 3 min before returning to normoxia. Following control microinjections with mock CSF, microinjections of either kainic acid or kynurenic acid were made and the fish exposed to normoxia and hypoxia as before. At the end of an experiment fish were killed with an overdose of anesthesia (MS-222). Heparin (0.2 ml, 5000 IU) was injected by cardiac puncture then fish were exsanguinated via the ventral aorta with approximately 60 ml of heparinized saline. Following exsanguinations, fish were perfused with 20 ml of 4% formaldehyde in phosphate buffered saline. Brains were removed and stored in 4% formaldehyde solution for anatomical studies. Serial transverse sections (20–40 μm) were cut and air-dried overnight on gelatine-coated glass slides. Sections were cover-slipped and viewed with a fluorescent microscope to confirm injection sites and estimate spread of injected material (see Sundin et al., 2003a). The effects of hypoxia and microinjections on heart rate and blood pressure were analyzed with a 2-way ANOVA. Specific effects (i.e. the effects of hypoxia after an injection) were analyzed using the least significant difference test for planned post hoc comparisons (P b 0.05). 3. Results 3.1. Mock CSF controls and response to hypoxia Control microinjections of mock CSF did not alter the cardiovascular effects of hypoxia (Figs. 2, 3). Fish responded as expected to hypoxia with a significant bradycardia after microinjections of mock CSF. The optic tectum lateral line lobe facial lobe vagal lobe obex
cerebellum
spinal cord
4th ventricle
injection sites
Fig. 1. Drawing of channel catfish brain (dorsal view) with major structures labeled and showing the approximate locations of microinjections (black dots).
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normoxia hypoxia
120
*
blood pressure (cm H2O)
40
100
heart rate (beats/min)
3
*
80 60 40 20
35 30 25 20 15 10 5
0 control
0
kainic acid
control
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Fig. 2. Mean cardiovascular responses to hypoxia after control (mock CSF) and kainic acid microinjections. Values are means ± S.E.M. (N = 6). * indicates statistically significant difference from normoxic control.
difference in blood pressure between normoxia and hypoxia was not statistically significant. 3.2. Kainic acid The cardiovascular effects of kainic acid microinjections are shown in Fig. 2 (N = 6). Heart rate in normoxia was 108 ± 6 beats min− 1 and decreased significantly to 82 ± 11 beats min −1 in aquatic hypoxia (P b 0.05). Following bilateral microinjection of kainic acid, heart rate decreased to 85 ± 2 beats min −1 (P b 0.05) in normoxia and did not change further during exposure to hypoxia (Fig. 2). Blood pressure was 33.3 ± 1.2 cm H2O in normoxia and was not affected by aquatic hypoxia, or by kainic acid and exposure to normoxia or hypoxia (Fig. 2). 3.3. Kynurenic acid The cardiovascular effects of kynurenic acid microinjections are shown in Fig. 3 (N = 8). Heart rate was 96 ± 5 beats min −1 in normoxia and hypoxia caused a significant bradycardia to 65 ± 6 beats min −1 (P b 0.05). After kynurenic acid injections normoxic heart rate was 76 ± 7 beats min −1 which was significantly lower than control heart rate. Following microinjection of kynurenic acid, exposure to aquatic hypoxia produced a significant bradycardia to 59 ± 9 beats min −1. Blood pressure was 24.4 ± 2.1 cm H2O in normoxia and was not affected by aquatic hypoxia, or by kynurenic acid and exposure to normoxia or hypoxia (Fig. 3).
3.4. Microscopy Microscopic examination of fixed brain slices confirmed that microinjections were made into the sensory region and not into underlying motor neurons. Spread of injections in the vertical plane was approximately 400 μm, independent of injection volume and between 650 and 1400 μm in the horizontal plane (Sundin et al., 2003a). 4. Discussion This study has shown that nGV microinjections of the ionotropic glutamate receptor antagonist kynurenic acid or chemical lesions of glutamatergic neurons with kainic acid significantly lower heart rate in normoxia. Furthermore, kainic acid, but not kynurenic acid, also blocks the bradycardia in response to aquatic hypoxia. These results suggest that central glutamatergic neurons exert a tonic excitatory control of HR in normoxia and glutamatergic receptors in the nGV play an integrative role in the cardiovascular response to aquatic hypoxia in the catfish. 4.1. Critique of methods First, in order to examine the role of centrally-microinjected neurochemicals on cardiovascular function in catfish, it was necessary to use lightly-anesthetized, spontaneously-breathing animals. There are some drawbacks to using MS 222 anesthesia, but anesthesia is necessary in
normoxia hypoxia 120
heart rate (beats/min)
100
* 80
*
+
60 40 20
blood pressure (cm H2O)
30 25 20 15 10 5 0
0 control
kynurenic acid
control
kynurenic acid
Fig. 3. Mean cardiovascular responses to hypoxia after control (mock CSF) and kynurenic acid microinjections. Values are means ± S.E.M. (N = 8). * indicates statistically significant difference from normoxic control. + indicates statistically significant difference from normoxic kynurenic acid.
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order to perform the microinjection experiments as we have done in this study. An alternative to this procedure would be to decerebrate the animals, thereby eliminating the need for anesthesia. However, previous studies have shown that decerebration causes unacceptable changes in blood pressure due to hemorrhage (Burleson and Smatresk, 1987; Sundin et al., 2003a,b). Resting heart rate in anesthetized, spontaneously-breathing catfish is considerably higher than either decerebrate or unanesthetized catfish, while anesthesia causes hypotension compared with unanesthetized fish. Anesthesia also attenuates the blood pressure and heart rate response to hypoxia (Burleson and Smatresk, 2000). The elevated heart rate under normoxic conditions is likely caused by the sympathetic cardiac stimulation by MS 222 (Smith, 1974) or by a direct excitatory effect on the central nervous system that occurs at low concentrations of MS 222 (Hedrick and Winmill, 2003). Another explanation for the high resting heart rate is that high water flow velocities associated with the assisted ventilation may have elevated resting heart rate via a mechanoreceptor reflex (Taylor and Butler, 1982; Barrett and Taylor, 1984). Despite these drawbacks, however, chemoreceptor stimulation by hypoxia produces cardioventilatory reflexes to aquatic hypoxia, albeit an attenuated response, compared with that of unanesthetized catfish (Burleson and Smatresk, 2000; Sundin et al., 2003a). Secondly, another critique is that both of the drugs used have effects on receptors and cells other than glutamatergic neurons and specific glutamate pathways. Kynurenic acid and its metabolites are now being shown to affect a number of different pathways. It stimulates the G-protein-coupled receptor GPR35 and, although controversial, may also inhibit α7 nicotinic acetylcholine receptors (Stone et al., 2013). Cell death by kainic acid releases a variety of excitatory amino acids, cytokines and inflammatory chemicals (Zhang and Zhu, 2011). All of these have potential to affect central control of cardiac function and could account for differences in the cardiovascular responses to kynurenic and kainic acid. Thirdly, despite negative immunohistochemical evidence for glutamate in the nGV, there were positive results for the NMDA receptor subunit 1 and both kynurenic and kainic acid had significant effects on cardiovascular variables. The lack of glutamate immunoreactivity contrasts with positive results in sculpin (Sundin et al., 2003b). It may be due to an error in protocol or processing of the tissues or variation between species but clearly needs to be re-examined. 4.2. Effects of kynurenic acid and kainic acid microinjections on heart rate and blood pressure Blocking glutamatergic transmission with both kainic and kynurenic acid resulted in a decrease in heart rate, suggesting that under normoxic conditions there is tonic excitatory glutamatergic input maintaining an elevated heart rate in lightly-anesthetized catfish. Loss of glutamatergic input by chemical lesions of glutamatergic neurons with kainic acid or blocking ionotropic glutamate receptors with kynurenate produced a similar degree of bradycardia under normoxic conditions. The results with glutamatergic antagonists were somewhat unexpected because microinjection of the agonist glutamate has been reported to result in bradycardia. For example, microinjection of glutamate into the nGV of shorthorn sculpin (Myoxocephalus scorpius) causes a reflex bradycardia (Sundin et al., 2003b). This reflex bradycardia is blocked by the NMDA receptor antagonist MK801 microinjected into the same glutamatesensitive site in the nGV suggesting that NMDA receptors mediate this response (Sundin et al., 2003b). In the present study, glutamate microinjections into the nGV of catfish produced no heart rate response (data not shown) and did not produce any ventilatory response in a previous study with catfish (Sundin et al., 2003a). The lack of cardioventilatory response is consistent with the finding that the nGV does not exhibit glutamate immunoreactivity in the catfish (Sundin et al., 2003a). Thus, the integration of cardioventilatory reflexes in the nGV appears to differ markedly between shorthorn sculpin and channel catfish.
A possible explanation for the results with kainic and kynurenic acid is that, in catfish, the nGV serves as an important region for integration of sensory input as well as integrating inputs from other regions of the brain. Because MS 222 anesthesia elevates heart rate, presumably through a sympathetic mechanism (Smith, 1974), the data in the present study indicate that glutamatergic blockade within the nGV probably reduces sympathetic outflow to the heart in catfish. Bradycardia induced by hypoxia or by glutamate microinjected into the nGV is primarily mediated by increased parasympathetic vagal input to the heart since it is blocked by atropine (Taylor et al., 1999; Sundin et al., 2003b). However, branchial nerve section in atropinized catfish fails to completely abolish the hypoxic bradycardia (Burleson and Smatresk, 1990) indicating that withdrawal of sympathetic tone to the heart may be involved in this reflex. Whether this would occur in unanesthetized catfish cannot be addressed by the present study. 4.3. Integration of cardiovascular reflexes in hypoxia Aquatic hypoxia normally evokes an increase in ventilation and a reflex bradycardia in fish owing to stimulation of chemoreceptors located on the gills (Nilsson, 1984; Burleson et al., 1992). Substantial evidence indicates that gill chemoreceptors are both externally and internally-oriented in order to respond to aquatic and blood oxygen status, respectively. Gill chemoreceptors are innervated by sensory branches of cranial nerves IX and X which terminate in the nGV to form the first-order relay for chemoreceptor-mediated reflexes. In teleosts, the nGV forms a continuous dorso-lateral column on either side of the 4th ventricle in the medulla, into which the sensory fibers from visceral structures terminate (Meek and Nieuwenhuys, 1998). Because the nGV represents the termination of gill chemoreceptor pathways in fish, central stimulation of this pathway by glutamate mimics the bradycardia and ventilatory increase evoked by peripheral chemoreceptor stimulation by hypoxia (Sundin et al., 2003a,b). In shorthorn sculpin, hypoxia or cyanide-evoked increases in ventilation are abolished by systemic injection of the NMDA receptor antagonist MK-801, but the bradycardia resulting from these stimuli is not blocked by MK-801 (Turesson and Sundin, 2003). This indicates that central glutamatergic pathways that modulate ventilatory and cardiovascular differ in response to peripheral chemoreceptor stimulation. In a previous study with catfish (Sundin et al., 2003a), microinjections of kainic acid into the nGV abolished breathing in normoxic conditions whereas kynurenic acid microinjections in the same area blocked the respiratory increase in hypoxia. In the present study, destruction of glutamatergic neurons in the nGV with kainic acid blocked the bradycardia in response to aquatic hypoxia, but kynurenic acid microinjections did not. A major difference between these two treatments is that while kainic acid destroys glutamatergic neurons and prevents the activation of both ionotropic and metabotropic glutamate receptors, kynurenic acid blocks only ionotropic receptors. This would suggest that metabotropic receptors may be involved in the integration of hypoxic reflexes within the nGV in catfish. Group I metabotropic glutamate receptors mediate cardiovascular reflexes within the NTS of urethane-anesthetized rats (Foley et al., 1999). Although control of heart rate by glutamatergic receptors within the nGV of catfish is indicated by the results of this study, the precise mechanisms and pathways of regulation are not clear. Our histological results (Sundin et al., 2003b) indicate that we were successful in blocking glutamatergic pathways within the sensory region of the medulla and did not evoke these responses by activation of underlying ventral motor areas. Because heart rate decreased in response to blockade of ionotropic receptors or loss of glutamatergic neurons within the nGV, this region represents a source or pathway for tonic glutamate release that maintains an elevated heart rate. This is consistent with the fact that glutamate microinjections in this region did not have any effect on heart rate in catfish (data not shown), whereas microinjections of glutamate in the nGV of shorthorn sculpin produced a bradycardia (Sundin
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et al., 2003b). As a comparison to the glutamate studies, intracerebroventricular injections of various neuropeptides in unanesthetized rainbow trout either had no effect or increased heart rate (Le Mével et al., 2012). These neuropeptides have been immunohistochemically localized to the same central cardioventilatory control areas and also work through G-protein-coupled receptors. It is not clear if the tonic glutamatergic input from the nGV occurs under the specific conditions of this study (i.e. light anesthesia), or if this is unique to catfish. Given the differences in cardioventilatory responses with microinjections of glutamatergic drugs into similar areas of the medulla between shorthorn sculpin (Sundin et al., 2003b: Turesson et al., 2010) and channel catfish (Sundin et al., 2003a, present study) under similar experimental conditions, it appears that there are substantial species differences in cardioventilatory regulation within the medulla. Acknowledgments This work was supported in part by grants from the National Institutes of Health (HL076205-01), the Swedish Research Council, Helge Axelson Johnson Stiftelse, Stiftelsen Längmanska Kulturfonden, and Wilhelm and Martina's Research Foundation. We thank Gunilla Rydgren for histological preparations of the brains and Adam Brooks for illustration of the catfish brain. References Axelsson, M., Fritsche, R., 1994. Cannulation techniques. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes, Analytical Techniques, vol. 3. Elsevier, Amsterdam, pp. 17–36. Barrett, D.J., Taylor, E., 1984. Changes in heart rate during progressive hypoxia in the dogfish Scyliorhinus canicula L.: evidence for a venous oxygen receptor. Comp. Biochem. Physiol. 78, 697–703. Burleson, M.L., Smatresk, N.J., 1987. The effect of decerebration and anesthesia on the reflex responses to hypoxia in catfish. Can. J. Zool. 67, 630–635. Burleson, M.L., Smatresk, N.J., 1990. Effects of sectioning cranial nerves IX and X on cardiovascular and ventilatory reflex responses to hypoxia and NaCN in channel catfish. J. Exp. Biol. 154, 407–420. Burleson, M.L., Smatresk, N.J., 2000. Branchial chemoreceptors mediate ventilatory responses to hypercapnic acidosis in channel catfish. Comp. Biochem. Physiol. A 125, 403–414. Burleson, M.L., Smatresk, N.J., Milsom, W.K., 1992. Afferent inputs associated with cardioventilatory control in fish. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish
5
Physiology. The Cardiovascular System, vol. XXIIB. Academic Press, New York, pp. 390–426. Foley, C.M., Vogl, H.W., Mueller, P.J., Hay, M., Hasser, E.M., 1999. Cardiovascular response to group I metabotropic glutamate receptor activation in NTS. Am. J. Physiol. 276, R1469–R1478. Hedrick, M.S., Winmill, R.E., 2003. Excitatory and inhibitory effects of tricaine (MS-222) on fictive breathing in isolated bullfrog brain stem. Am. J. Physiol. 284, R405–R412. Hoffman, A., Brazil Romero, S.M., de Oliveira, L.M., 1993. Agonistic behavior and its cardiovascular components elicited by microinjection of L-glutamic acid into the basal midbrain of the toad Bufo paracnemis. Brain Behav. Evol. 41, 316–325. Kanwal, J.S., Caprio, J., 1987. Central projections of the glossopharyngeal and vagal nerves in the channel catfish, Ictalurus punctatus: clues to differential processing of visceral inputs. J. Comp. Neurol. 264, 216–230. Le Mével, J.C., Lancien, F., Mimassi, N., Conlon, J.M., 2012. Brain neuropeptides in central ventilatory and cardiovascular regulation in trout. Front. Endocrinol. 3, 124. http:// dx.doi.org/10.3389/fendo.2012.00124. Lin, H.-C., Wan, F.-J., Tseng, C.-J., 1999. Modulation of cardiovascular effects produced by nitric oxide and ionotropic glutamate receptor interaction in the nucleus tractus solitarii of rats. Neuropharmacology 38, 935–941. Meek, J., Nieuwenhuys, R., 1998. Holosteans and teleosts. In: Nieuwenhuys, R., Ten Donkelaar, H.I., Nicholson, C. (Eds.), The Central Nervous System of Vertebrates, vol. 2. Springer, Berlin, pp. 759–937. Nilsson, S., 1984. Innervation and pharmacology of the gills. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. 10A. Academic Press, New York, pp. 185–227. Ohtake, P.J., Torres, J.E., Gozal, Y.M., Graff, G.R., Gozal, D., 1998. NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat. J. Appl. Physiol. 84, 853–861. Smith, D.G., 1974. Sympathetic cardiac stimulation in Bufo marinus under MS-222 anesthesia. Am. J. Physiol. 226, 367–370. Stone, T.W., Stoy, N., Darlington, G.L., 2013. An expanding range of targets for kynurenine metabolites of tryptophan. Trends Pharmacol. Sci. 34, 136–143. Sundin, L., Turesson, J., Burleson, M., 2003a. Identification of central mechanisms vital for breathing in the channel catfish, Ictalurus punctatus. Respir. Physiol. Neurobiol. 138, 77–86. Sundin, L., Turesson, J., Taylor, E.W., 2003b. Evidence for glutamatergic mechanisms in the vagal sensory pathway initiating cardiorespiratory reflexes in the shorthorn sculpin Myoxocephalus scorpius. J. Exp. Biol. 206, 867–876. Taylor, E.W., Butler, P.J., 1982. Nervous control of heart rate: activity in the cardiac vagus of the dogfish. J. Appl. Physiol. 43, 1330–1335. Taylor, E.W., Jordan, D., Coote, J.H., 1999. Central control of the cardiovascular and respiratory systems and their interactions in vertebrates. Physiol. Rev. 79, 855–916. Turesson, J., Sundin, L., 2003. N-methyl-D-aspartate receptors mediate chemoreflexes in the shorthorn sculpin Myoxocephalus scorpius. J. Exp. Biol. 206, 1251–1259. Turesson, J., Johansson, M., Sundin, L., 2010. Involvement of non-NMDA receptors in central mediation of chemoreflexes in the shorthorn sculpin, Myoxocephalus scorpius. Respir. Physiol. Neurobiol. 172 (3), 83–93. Villar-Cerviño, V., Barreiro-Iglesias, A., Fernández-lópez, B., Mazan, S., Rodicio, M.C., Anadón, R., 2013. Glutamatergic neuronal populations in the brainstem of the sea lamprey, Petromyzon marinus: an in situ hybridization and immunocytochemical study. J. Comp. Neurol. 521, 522–557. http://dx.doi.org/10.1002/cne.23189. Zhang, X.M., Zhu, J., 2011. Kainic acid-induced neurotoxicity: targeting glial responses and glia-derived cytokines. Curr. Neuropharmacol. 9, 388–398.