Involvement of non-NMDA receptors in central mediation of chemoreflexes in the shorthorn sculpin, Myoxocephalus scorpius

Respiratory Physiology & Neurobiology 172 (2010) 83–93

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Involvement of non-NMDA receptors in central mediation of chemoreflexes in the shorthorn sculpin, Myoxocephalus scorpius J. Turesson ∗ , M. Johansson, L. Sundin Department of Zoology, Göteborg University, Box 463, S-405 30 Gothenburg, Sweden

a r t i c l e

i n f o

Article history: Accepted 19 March 2010 Keywords: Teleost Hypoxia Respiration Ventilation Cardiovascular Nucleus tractus solitarius Glutamate AMPA NMDA Kainate Ionotropic

a b s t r a c t NMDA receptors mediate hypoxia-induced ventilatory frequency and blood pressure increases in fish. Here we continue to resolve whether non-NMDA receptors participate in chemoreflexes. Shorthorn sculpins, instrumented for cardiorespiratory measurements, were kept unrestrained or positioned in a stereotaxic frame. Chemoreflexes were elicited (hypoxia/NaCN-induced) before/after administration of either the specific AMPA receptor antagonist, GYKI52466 (systemically), or the specific kainate receptor antagonist, UBP293 (microinjections into fourth ventricle). Immunohistochemistry was performed on medullary cross-sections to identify non-NMDA receptor subunits in the chemoreflex-pathway. Kainate receptors mediate the chemoreflex-mediated increase in ventilation amplitude, since the response was abolished by UBP293. GYKI52466 attenuated the ventilatory frequency increase, and induced more regular breathing patterns and higher heart rate in both normoxic and hypoxic conditions, suggesting that AMPA receptors also partake in cardiorespiratory control. This together with immunohistochemical findings of both AMPA and kainate receptor subunits in the chemoreflex-pathway, show that non-NMDA receptors play a role in both chemoreflex-activation and normoxic cardiorespiratory regulation in fish. © 2010 Published by Elsevier B.V.

1. Introduction The importance of the excitatory amino acid (EAA) glutamate and the N-methyl-D-aspartate (NMDA) receptor in central control of ventilatory and cardiovascular oxygen chemoreflexes has been well established in mammals (Mizusawa et al., 1994; Haibara et al., 1995; Lin et al., 1996; Ohtake et al., 1998) and in fish (Sundin et al., 2003a; Turesson and Sundin, 2003; Turesson et al., 2006). However, information on the potential role of non-NMDA receptors (␣-amino-3-OH-5-methyl-4-isoxazole-propionic-acid (AMPA) and kainate) in vertebrate respiratory control is limited. The first integration of sensory information from the oxygen chemoreceptors occurs when their afferents release neurotransmitters such as the excitatory amino acid glutamate into the nucleus tractus solitarius (NTS). Here glutamate activates ionotropic glutamate receptors and cardiorespiratory reflexes are produced (Van Giersbergen et al., 1992; Perrone, 1981; Mizusawa et al., 1994; Saha et al., 1995; Sykes et al., 1997). In fish, the major part of the oxygen chemoreceptors initiating cardiorespiratory responses is situated in the orobranchial cavity (Hughes and Shelton, 1962; Saunders and Sutterlin, 1971; Sundin et al., 1999, 2000; Milsom et al., 2002). The majority of these receptors are

∗ Corresponding author at: Department of Zoophysiology, Göteborg University, Box 463, S-405 30 Göteborg, Sweden. Tel.: +46 31 786 3697; fax: +46 31 786 3807. E-mail address: [email protected] (J. Turesson). 1569-9048/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.resp.2010.03.019

innervated by the ninth (glossopharyngeal) and 10th (vagal) cranial nerves, where the terminal field (sensory area) in the fish medulla is less defined but the equivalent to the NTS in mammals (Sundin et al., 2003b). In fish it appears that only the hypoxia-induced increase in ventilation rate is mediated via NMDA receptors, while the concurrent increase in amplitude is not (Turesson and Sundin, 2003). However, both of the ventilatory reflex responses are abolished after application of kynurenic acid (a broad-spectrum ionotropic glutamate receptor antagonist) into the vagal sensory area in the channel catfish (Sundin et al., 2003a). Therefore, we proposed that either AMPA or kainate receptors or both of the non-NMDA receptors are mediating the hypoxia-activated increase in ventilation amplitude in fish (Sundin et al., 2003a). Corroborating our suggestion is the finding that kynurenic acid (via intracerebroventricular injection) reduced the hypoxia-induced increase in tidal volume in rats (de Paula and Brancho, 2004). Strengthening the results from the pharmacological experiments, immunocytochemical studies have identified ionotropic non-NMDA receptor subunits in the whole rostro-caudal length of the mammalian NTS (Robinson and Ellenberger, 1997; Ambalavanar et al., 1998), so far only the presence of NMDA receptors have been established in the vagal sensory area in the shorthorn sculpin (Turesson and Sundin, 2003). Apart from raised ventilation in response to a hypoxic exposure, fish generally also display a bradycardia mediated by the cholinergic inhibition of the heart, as well as hypertension through

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sympathetic activation of peripheral ␣-adrenoceptors (Randall, 1982; Nilsson, 1983; Taylor, 1985; Burleson and Smatresk, 1990; Fritsche, 1990; Fritsche and Nilsson, 1990). It appears that central NMDA receptors are mediating the hypoxic pressor response (Turesson and Sundin, 2003), but more information on central glutamatergic regulation of hypoxic cardiovascular reflexes in fish is needed to establish ionotropic glutamate receptors as vital for the central mediation of cardiovascular chemoreflexes as has been done in mammals (Perrone, 1981; Miyawaki et al., 1996; Mizusawa et al., 1994; Haibara et al., 1995; Ohtake et al., 1998). MK801, an NMDA receptor antagonist, produces very different irregular breathing patterns in mammals (anesthetized), such as inspiratory prolongation (Connelly et al., 1992; Ling et al., 1994) and an increase in ventilation frequency (Harris and Milsom, 2001). Similarly, in normoxic shorthorn sculpins, we have observed that the breathing pattern in the presence of MK801 substantially alters and becomes more irregular. The new types of MK801induced breathing patterns include continuous breathing but at an increased frequency, frequency cycling, and episodic breathing (Turesson and Sundin, 2003). Interestingly the MK801-induced irregular breathing patterns become more regular during hypoxic conditions. That non-NMDA receptors are also important in breathing pattern generation in mammals was shown when GYKI52466 and NBQX were intravenously infused into cats. After non-NMDA receptor blockade the spontaneous discharge of respiratory neurons was reduce or even abolished (Pierrefishe et al., 1994). Consequently, as MK801 appears to play a role in shaping breathing patterns in shorthorn sculpins, at least in normoxic conditions, we speculate whether other ionotropic glutamate receptors are involved as well. The main objective of this study was to determine the putative role of non-NMDA receptors in the glutamatergic mediation of cardiorespiratory chemoreflexes in fish. This was accomplished using both a specific AMPA receptor antagonist (GYKI52466), and a specific kainate receptor antagonist (UBP296). In addition, an immunohistochemical analysis of non-NMDA receptor subunits in the vagal sensory area was also performed. A second objective was to determine and compare the roles of non-NMDA and NMDA receptors in regulating breathing patterns. Therefore, we analyzed the interbreath intervals extracted from present raw data and previously obtained NMDA raw data (Turesson and Sundin, 2003). 2. Material and methods 2.1. Animals Shorthorn sculpins (Myoxocephalus scorpius) were caught outside the Swedish West Coast, brought back to the department of Zoophysiology at Göteborg University, and maintained as previously described (Turesson and Sundin, 2003). The GYKI52466 treatment group was experimented on in May, the UBP296 treatment group in April and the MK801 treatment group (data from Turesson and Sundin, 2003) in October, March and April. All animal experiments were approved by the local ethical committee in Gothenburg (permit no. 331-2002). 2.2. Systemic injections of GYKI52466, an AMPA receptor antagonist 2.2.1. Surgical procedure Shorthorn sculpins (N = 9, ranging in body mass between 161 and 344 g) were placed in an anesthetic solution containing MS222 (3-amino-benzioc acid ethylester methanesulphfonate, obtained from Sigma Aldrich, 100–150 mg l−1 saltwater) and transferred to

an operating table when spontaneous breathing ceased. During the whole surgery cooled (10 ◦ C) aerated saltwater with a lower MS222 concentration (50–75 mg l−1 saltwater) was passed over the gills. To facilitate ventral aortic pressure (Pva ) measurements, the fish were instrumented with a polyethylene cannula (PE50, tipped with a thinner (PE10) cannula) into the afferent branchial artery of the third gill arch (Axelsson and Fritsche, 1994). A second cannula (PE 90) was inserted into the branchial operculum for ventilation measurements. After surgery was completed, the fish were transferred to the experimental channel and were given at least a day to recover. 2.2.2. Experimental set-up As the antagonist was systemically injected (via the branchial cannula, GYKI52466 can easily pass the blood–brain barrier), the fish remained awake and could to some extent swim around in the chamber. The branchial cannula was filled with heparinized (100 IU ml−1 ) 0.9% NaCl. The branchial and opercular cannulae were connected to pressure transducers previously calibrated against a static water column. The signals from the transducers, amplified via a Grass lowlevel D.C. amplifier (Grass Inc. model 7P122B, Quincy, USA), were continuously sampled at 20 Hz using a PowerLab/16 SP 16 channel recorder (AD Instruments, Castle Hill, Australia) and finally transferred to a data acquisition software program (PowerLab version 5.0.2, AD Instruments, Castle Hill, Australia). 2.2.3. Experimental protocols The experiments started with a 5 min long resting period when the cardiorespiratory variables were stable. Thereafter the fish were subjected to 10 min of hypoxia, where the PO2 gradually dropped from 21 kPa to a minimum level of 5 kPa within 5 min (see Fig. 1E). For the remainder of the hypoxic period the PO2 was kept constant at the minimum level. The nitrogen bubbling was then turned off and the water returned to normoxia again by bubbling with air. The fish were allowed to recover until the cardiorespiratory variables had stabilized to pre-hypoxic levels, usually after 10–15 min. Following the control hypoxic exposure the fish were systemically injected with the specific AMPA receptor non-competitive antagonist GYKI52466 (obtained from Sigma Aldrich, 12 mg kg−1 , a similar dose as used in mammalian studies, Pierrefiche et al., 1994; 12 mg kg−1 , a similar dose as used in mammalian studies, Colak et al., 2003), and after approximately 60–90 min, when the cardiorespiratory variables had stabilized, the hypoxic exposure was repeated. GYKI52466 was dissolved in DMSO in 10% 0.1 mM HCl. To verify that the vehicle did not have an effect on the fish another group of fish (N = 4, ranging in body mass between 222 and 336 g) were treated with the vehicle per se and exposed to hypoxia as described above. No significant changes in either resting values or the hypoxic responses were seen before or after the vehicle injection. 2.3. Microinjections experiments of UBP296, a kainate receptor antagonist 2.3.1. Surgical procedure Another group of fish (N = 7), body mass 161–344 g, were equipped with ventral aortic cannulas as described in Section 2.2.1. A PE90 cannula was inserted through the snout for administration of sodium cyanide (NaCN). After initial surgery was completed, the fish were moved into a holding chamber and were given at least 24 h to recover. On the experimental day, the fish were again anaesthetized in MS222 and a small rare-earth magnet was glued onto the operculum unilaterally for ventilation measurements. The fish were

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procedure the top of the head was prevented from submersion by regulating the water level using an adjustable standpipe inside the chamber. After surgery the amount of MS222 in the water was reduced to 40–50 mg l−1 , and when the mouth tube was withdrawn unrestricted, spontaneous breathing movements returned. Finally, the fish was allowed to stabilize from handling for at least an hour or until cardiovascular and ventilatory variables were stable. 2.3.2. Experimental set-up The branchial cannula was connected to a pressure transducer. Ventilation was measured via a Hall effect transducer (Honeywell) by recording the movements of the rare-earth magnet attached to the operculum. The signals from the pressure transducer and the Hall effect transducer were amplified and sampled with a frequency of 20 Hz using a data acquisition software program (Labview version 5.0, National Instruments, Austin, USA). See Section 2.2.2 for more details. A single-barrel glass pipette (tip size 10–15 ␮m) was connected to a pressure injector (Harvard apparatus, Medical Systems, Research Products), and used to make the injections into the fourth ventricle. To facilitate more exact injections, the glass pipette was fixed in a micromanipulator unit (SM15 equipped with a base SM15M) mounted on the stereotaxic frame. 2.3.3. Experimental protocol The experimental protocol started with a 2 min long resting period followed by a NaCN injection (0.5 ml, 1.0 mg/ml) into the respiratory water through the snout cannula. To study the involvement of kainate receptors in the chemoreflex-activation, the fish were injected with UBP296 (250–350 nl, obtained from Tocris Bioscience and dissolved in 20% DMSO in 0.9% NaCl) into the forth ventricle. Other kainate receptor blockers have been reported to show poor brain penetration (More et al., 2004). Even though we found no information about the UBP296 ability to penetrate the brain we still decided to choose microinjection as the technique for administration of the antagonist. The injection was made approximately 40 min after the first NaCN injection. After the UBP296 injection, the animals were left for approximately 80 min before the NaCN injection was repeated. The volumes of the injections were determined by observing the movement of the meniscus inside the pipette with a calibrated eyepiece micrometer placed on a microscope. To take into the account the potential effect of the UBP296 vehicle (20% DMSO, 0.9% NaCl) on the chemoreflex-activation, the vehicle (250–350 nl) was injected into the fourth ventricle in each fish before making the first NaCN injection through the snout. Fig. 1. Summary of hypoxia-elicited cardiorespiratory reflexes. Hypoxic effects on ventilation rate (fV ) (A), ventilation amplitude (AMPV ) (B), heart rate (fH ) (C) and ventral aortic pressure (Pva ) (D) in control fish (♦) and in GYKI52466 (AMPA receptor antagonist) treated fish (). Changes in partial oxygen tension (PO2 ) in the respiratory water during hypoxia (E). Values shown are means + S.E.M. (N = 9). (*) Indicates a statistical significant difference (p < 0.05) from normoxic values. (#) Indicates a statistical significant difference (p < 0.05) between control and GYKI52466 treated fish.

then transferred to the experimental chamber and the head was rigidly held by clamps (attached to a modified stereotaxic frame, Narishige model SN-2N) positioned on the orbital ridges. Through a tube inserted into the mouth recirculating saltwater with MS222 (aerated, 10 ◦ C, 50–75 mg MS222 l−1 saltwater) was passed over the gill curtains keeping the fish anaesthetized and well oxygenated. While still under deep anesthesia limited craniotomy (hole diameter: approximately 1 cm) was performed to expose the back of the cerebellum, the fourth ventricle and the medulla. During this

2.4. Calculations and statistical analysis From the recorded PVA and ventilation signals, heart rate (fH ), ventilation amplitude (AMPV ) and ventilation frequency (fV ) were calculated using a Labview-based program. Mean values were created at 10 s intervals and plotted as graphs. To determine the effects of GYKI52466 and UBP296 in chemoreflex-mediation, and thus the involvement of AMPA and kainate receptors, delta values (maximum/minimum value – resting value) for each cardiovascular and ventilatory variable in both untreated (control) and GYKI52466 or UBP296 treated fish were compared using Student’s paired t-test. The results are presented as mean ± S.E.M. A value of p < 0.05 was considered significant. To establish whether UBP296 and GYKI52466 respectively affected the cardiovascular and ventilatory systems during normoxia, resting values from the period just before the chemoreflexactivation (see Figs. 1 and 2) before and after antagonist treatment were compared with each other using Student’s paired t-test.

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For glutamatergic breathing pattern variability, interbreath intervals (during normoxia; the last 5 min before N2 was turned off; and the 2 min following NaCN injection); before and after GYKI52466, MK801 (raw data from a previous study, Turesson and Sundin, 2003) and UBP296 treatment respectively) were analyzed with HRV Analysis Software 1.1 for Windows (developed by The Biomedical Signal Analysis Group, Department of Applied Physics, University of Kuopio, Finland). The HRV analysis transform the data from each individual fish into Poincare plots that graph the interval between two breaths (n) versus the interval between the subsequent two breaths (n + 1). From each individual plot the HRV analysis program then calculates SD1 and SD2 values from an ellipse that has been mathematically fitted to the distribution of points (see Fig. 3). The SD1 value describes short-term variability, and the SD2 value long-term variability (Brennan et al., 2001). For example, if both SD1 and SD2 are very small, every breath last almost exactly as long as the previous one and breathing is thus very regular. If the SD1 is small but SD2 is large, it indicates that there is an infinitesimally small reduction in interbreath interval from one breath to another, which over time results in a faster breathing frequency. Finally, if both SD1 and SD2 are large, there is variability between two consecutive breaths and the whole set of analyzed interbreath intervals vary over a large range of different values. Statistical analysis was performed using two-way repeated measures ANOVA with the respective SD1 or SD2 value from each fish as the repeated measure and oxygen tension (normoxia, hypoxia) and treatment (before antagonist, after antagonist) as categorical variables. In each case post hoc comparisons were made using Student’s paired t-test. When a data set was use repeatedly in the post hoc analysis Bonferroni–Holm’s correction was made. The HRV analysis was performed on each fish individually and Figs. 3–5 summarizes the interbreath intervals from each antagonist treated and untreated fish group. 2.5. Immunohistochemical experiments The presence of non-NMDA receptor subunits GluR2/3 and GluR5/6/7 in nerve fibres and on cell bodies in the medulla oblongata was established using single-labelling immunohistochemistry (antisera listed in Table 1). Seven animals were sacrificed with an overdose of MS222 (300–400 mg l−1 seawater) and the medulla was removed. After fixation in 4% paraformaldehyde overnight, the tissues were then rinsed in phosphate buffer (PBS, 0.9% NaCl) for 30 min. Prior to embedding in mounting medium (Tissueteck) and freezing in isopentane (cooled in liquid nitrogen), the tissues were cryoprotected in PBS-sucrose solution (0.9% NaCl, 30% sucrose) overnight. Using a cryostat (Cryo-Star HM 560M. Microm, Walldorf, Germany), cross-sections of the medulla (12 ␮m thick) were cut and mounted on gelatine-coated slides. The tissue sections were preincubated in Normal Donkey Serum (NDS, Dilution 1:10, to prevent unspecific binding by the secondary antiserum) for 30–60 min before the primary antibody incubation proceeded. The slides were incubated with primary antisera against the AMPA receptor subunits GluR2/3 or the kainate receptor subunits GluR5/6/7 Table 1 Primary and secondary antisera used in the immunohistochemical experiments. Code

Fig. 2. Summary of chemoreflex-activation before and after UBP296 (kainate receptor antagonist) treatment. NaCN-induced changes in ventilation rate (fV ) (A), ventilation amplitude (AMPV ) (B), heart rate (fH ) (C) and ventral aortic pressure (Pva ) (D) in control fish (♦) and in UBP296 () treated fish. Values shown are means + S.E.M. (N = 7). (*) Indicates a statistical significant difference (p < 0.05) from resting values. (#) Indicates a statistical significant difference (p < 0.05) between control and UBP296 treated fish.

Host

Dilution

Source

Primary antiserum raised against G5665 GluR 2/3* MAB379 GluR 5/6/7#

Rabbit Mouse

1:50 1:1000

SIGMA Chemicon

Secondary antiserum raised against DaR-CY3, rabbit IgG 711-165-152 DaM-FITC, mouse IgG 715-095-150

Donkey Donkey

1:800 1:100

Jackson Jackson

* #

Subunits of the AMPA receptor. Subunits of the Kainate receptor.

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Fig. 3. Poincare’ plots of hypoxia-induced changes in interbreath intervals (IBIs) before (−MK801) and after (+MK801) MK801 treatment (N = 7). The points in the scatter show all individual values from all fishes plotted together. IBI n is plotted versus the following IBI n + 1. SD1 and SD2, shown on the upper left panel, are parameters that quantify and describe the spread of the points. Similar PO2 regime as in Fig. 1.

Fig. 4. Poincare’ plots of hypoxia-induced changes in interbreath intervals (IBIs) before (−GYKI52466) and after (+GYKI52466) GYKI52466 treatment (N = 9). The points in the scatter show all individual values from all fishes plotted together. IBI n is plotted versus the following IBI n + 1. For PO2 changes see Fig. 1.

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Fig. 5. Poincare’ plots of NaCN-induced changes in interbreath intervals (IBIs) before (−UBP296) and after (+UBP296) UBP296 treatment (N = 7). The points in the scatter show all the individual values from all fishes plotted together. IBI n is plotted versus the following IBI n + 1.

for 24 h at room temperature. Excess of primary antiserum was removed in three steps of washing in PBS (2.0% NaCl) for 10 min each, followed by incubating the slides with the secondary antiserum DaR-CY3 or DaM-FITC for 60 min. After final rinse in PBS as previously described, the slides were cover-slipped in carbonate buffered glycerol. Finally, the slides were examined through a fluorescence microscope (Olympus BX 60, Olympus Optical Co. Ltd., Tokyo, Japan) and photographed with a Nikon digital camera DMX 1200. 3. Results 3.1. Cardiovascular and respiratory chemoreflexes 3.1.1. GYKI52466 treated fish Both fV and AMPV increased in response to the hypoxic exposure (Fig. 1A–B). GYKI52466 pre-treatment significantly reduced the peak fV increase but the remaining increase over normoxic levels was still significantly different from resting levels. In contrast to the significant attenuation of the frequency response, GYKI52466 did not affect the hypoxia-induced increase in AMPV . Unlike previous results (Turesson and Sundin, 2003) the hypoxia-induced bradycardia was absent in this group of sculpins (Fig. 1C). The blood pressure showed a gradual increase from the onset of hypoxia and reached a maximum level at the end of the hypoxic period and this increase was comparable to previous results (Fig. 1D). After GYKI52466 treatment the animals displayed a hypoxic bradycardia and the pressor response was abolished (Fig. 1C–D). 3.1.2. UBP296 treated fish The responses from NaCN injections mimicked hypoxia-induced chemoreflexes, including significant increase in ventilation (both in

frequency and amplitude, Fig. 2A–B), and marked bradycardia and decreased Pva (Fig. 2C–D). 80 min after UBP296 treatment the NaCN did not stimulate ventilation, both the frequency and the amplitude response was significantly blocked (Fig. 2A–B). The marked bradycardia and the concurrent decrease in blood pressure were unaffected by the UBP treatment (Fig. 2C–D). 3.2. Resting levels GYKI52466 significantly increased the resting level of heart rate and ventral aortic pressure (Table 2). In addition, GYKI52466 significantly decreased the resting ventilation amplitude, while resting levels of ventilation frequency was unaffected by the antagonist. UBP296 did not significantly affect any of the cardiorespiratory resting levels (Table 3). 3.3. Breathing patterns The poincare’ plots revealed that during normoxia, the interbreath intervals in untreated fish were different from the intervals after treatment with the respective antagonists in all three treatment groups (MK801, GYKI52466 and UBP296) (Figs. 3–6). The Table 2 Mean resting values of cardiovascular and ventilatory variables before (control) and after GYKI52466 treatment. Values are shown as mean ± S.E.M. (N = 9). Variable

Control

GYKI52466

fV (breaths min−1 ) AMPV (kPa) PVA (kPa) fH (beats min−1 )

10.2 ± 1.7 0.12 ± 0.01 2.9 ± 0.1 22.4 ± 1.7

13.6 ± 0.8 n.s. 0.08 ± 0.01* 3.4 ± 0.2* 36.3 ± 1.9*

n.s., no significance. * Statistical significant difference in resting value after GYKI52466 treatment.

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Fig. 6. Histograms of SD1 and SD2 from MK801, GYKI52466 and UBP296 treated fish. (*) Indicates a statistical significantly difference between normoxic period versus exposure to hypoxia or NaCN (for the UBP296 treated fish), before or after antagonist. (#) Indicates a statistical significant difference between untreated versus antagonisttreated fish during normoxia or hypoxia, or NaCN (for the UBP296 treated fish). p < 0.05 for MK801 treated fish; p < 0.025 and 0.05 after Bonferroni–Holm’s correction for GYKI52466 and UBP296 treated fish. Nota bene, the scale in the UBP296 bar charts is different from the MK801 and GYKI52466 bar charts.

plots also reveal that MK801 and GYKI52466 treatments affect interbreath intervals more than UBP296. MK801 increased the irregularity of breathing during normoxia, while GYKI52466 promoted regular breathing (Figs. 3–4, 6). 3.4. Immunohistochemical experiments Single labelling with the antisera listed in Table 1 was used to identify AMPA receptor subunits (GluR2/3) and kainate receptor subunits (GluR5/6/7) within the vagal sensory area, the part of NTS where vagal afferent fibers terminate, as well as the vagal motor area. The immunohistochemical results disclosed strong GluR2/3like immunoreactivity (IR) in fibres and cell bodies (ranging in size 11–15 ␮m) throughout the whole vagal sensory column (Fig. 7A–B, Table 3 Mean resting values of cardiovascular and ventilatory variables before (control) and after UBP926 treatment. Values are shown as mean ± S.E.M. (N = 7). Variable

Control

UBP296

fV (breaths min−1 ) AMPV (arbitrary units) fH (beats min−1 ) PVA (kPa)

33.8 ± 2.0 0.57 ± 0.1 51.2 ± 2.5 0.29 ± 0.06

32.5 ± 2.3 n.s. 0.56 ± 0.1 n.s. 49.6 ± 2.9 n.s. 0.33 ± 0.06 n.s.

n.s., no significance.

D). The cell bodies in the vagal motor area showed both GluR2/3 and GluR5/6/7-like IR (Fig. 7B–C). No GluR2/3 or GluR5/6/7 -like IR was seen in blank tests where the primary antiserum was excluded, showing that the secondary antiserum bound specifically to the primary antiserum (Fig. 7B, left side). 4. Discussion Information on the potential role of non-NMDA receptors in respiratory control in vertebrates is limited. Whitney et al. (2000) showed a modest role for non-NMDA receptors in regulating breathing patterns in neonatal rats and one earlier study using lamprey brain preparations has shown a role for non-NMDA receptors in neurotransmission in the respiratory network (Bongianni et al., 1999). Here is the first report on non-NMDA receptor involvement in chemoreflex responses and breathing pattern formation in vivo in a teleost species. 4.1. The role of non-NMDA receptors in chemoreflex-mediated ventilatory responses It is clear that ionotropic glutamate receptors are involved in chemoreflex transmission in fish, since kynurenic acid (a broad spectrum ionotropic glutamate receptor antagonist) applied into

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Fig. 7. Micrographs showing cross-sections of the medulla, immunostained with GluR2/3 antiserum. (A) GluR2/3-like IR in the commissural part at position I (defined in D). Asterisks indicate interneurons. (B) GluR2/3-like IR in vagal afferent nerve fibers leading into the vagal sensory area and in numerous interneurons (asterisks) at position II (defined in D). Left side of micrograph (B) is a control where the primary antiserum was excluded. Arrow indicates GluR2/3-like IR in vagal motor neurons. (C) GluR5/6/7-like IR in neurons in the vagal motor area. (D) Schematic drawing (modified from Sundin et al., 2003) showing the medulla and the two positions (I and II) from where the micrographs were collected. Scale bars are 100 ␮m.

the viscerosensory area of the brainstem in channel catfish, prevents the ventilatory augmentation produced by hypoxia (Sundin et al., 2003a). Moreover, in shorthorn sculpins, the NMDA receptor antagonist MK801 only inhibits the hypoxia-induced increase in ventilation frequency but not the elevation in amplitude, why mediation by non-NMDA receptors is likely for amplitude changes (Turesson and Sundin, 2003). In this study, to determine if nonNMDA receptors partake in the hypoxic chemoreflex, the specific kainate receptor antagonist UBP296 was directly applied into the 4th ventricle of the medulla in one group of shorthorn sculpins. As this treatment resulted in decrease of the NaCN-induced elevated amplitude, it indeed suggests that non-NMDA receptors are vital for the production of an amplitude response in this species. GYKI52466, a highly specific AMPA receptor antagonist, was injected systemically into a second group of fish to further disentangle whether both non-NMDA receptors mediates the chemoreceptor-activated amplitude response. Interestingly, similar to the previously shown inability of MK801 (Turesson and Sundin, 2003), GYKI52466 could not significantly reduce the hypoxia-induced amplitude increase. Furthermore, supporting this suggestion is that kainate administered into the fourth ventricle increases ventilation amplitude in fetal sheep (Bissonnette et al., 1997). Altogether, this indicates that the regulation of ventilation by glutamatergic mechanisms show a high level of complexity as there are specific glutamate receptor types for each ventilatory reflex modality, i.e. NMDA receptors for ventilation frequency and kainate receptors for modulating the ventilation amplitude. Unlike the inability of GYKI52466 to reduce the amplitude, this AMPA receptor antagonist, as well as the kainate receptor antagonist, significantly reduced the hypoxia or NaCN (for UBP293)induced frequency increase. Given that the frequency augmentation during hypoxia is totally dependent on NMDA receptors in

shorthorn sculpins (Turesson and Sundin, 2003), the reduction in frequency observed after either AMPA or kainate receptor inhibition probably reflects the NMDA receptors’ dependence on membrane depolarization to become functional (Monaghan et al., 1989). Depolarization of the membrane via e.g. AMPA and/or possibly kainate receptors leads to removal of the voltage-gated Mg2+ block that is present in NMDA receptors at resting potential. 4.2. The role of non-NMDA receptors in cardiovascular control during different oxygen levels It has been shown that non-NMDA receptors mediate afferent synaptic transmission (Andresen and Yang, 1990). Also, AMPA microinjected into the NTS produces a bradycardia mediated by parasympathetic excitation (Takaoka et al., 2003). Furthermore, non-NMDA receptors mediate sympathetic reflexes in rats (Hong and Henry, 1992; Miyawaki et al., 1996; Li et al., 2001). Therefore, it is evident that there exists a role for non-NMDA receptors in the cardiovascular control in mammals. Thus, in addition to disentangle the non-NMDA receptor involvement in respiratory chemoreflex responses, blood pressure and heart rate was concurrently monitored in the present study. 4.2.1. AMPA receptors and control of heart rate In contrast to the finding of an AMPA mediated parasympathetic excitation by Takaoka and Machado (2003) is that GYKI52466 treatment disclosed a significant hypoxia-induced reflex bradycardia that was not seen in the untreated animals. In the UBP296 treatment group however, the untreated fish showed the typical chemoreflex bradycardia, but this response was still as prominent after the blockade. This suggests that non-NMDA receptors do not mediate a parasympathetic excitation in fish during chemoreflex-

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activation. However, it is still possible that AMPA directly applied into the NTS of shorthorn sculpins will affect the heart through a parasympathetic activation and, that other reflexes such as the baroreflex might depend on the involvement of AMPA receptors as has been shown in rats by Andresen and Yang (1990), and Zhang and Mifflin (1998). Paradoxically to the possible absence of a role in parasympathetic reflex on the heart rate, but in line with the finding of Takaoka and Machado (2003), a prominent effect of GYKI52466 on awake fish was the increase in resting heart rate from approximately 22 to 36 beats min−1 . From this result it appears that the resting cholinergic tonus on the heart in shorthorn sculpins depends on a parasympathetic excitation via AMPA receptors, possibly located in the brainstem. However, as AMPA receptor subunits have been found within the heart (Gill et al., 1998), and as the GYKI52466 was injected systemically, it is possible that there is a glutamatergic modulation of the parasympathetic system present in the heart itself acting via AMPA receptors. 4.2.2. AMPA receptors and control of blood pressure The systemic hypertension produced by hypoxia in fish is mediated via ␣-adrenoceptors (Fritsche and Nilsson, 1993), and some contribution from the gills occurs via parasympathetic muscarine receptors (Sundin and Nilsson, 1997). In this study the total hypertension was abolished when the animals were pre-treated with GYKI52466, which suggest that the activation of the sympathetic component of the vascular reflex is mediated by non-NMDA receptors. The same results but with pre-treatment with MK801 have been shown earlier and it was argued that sympathetic component of the vascular reflex is mediated by NMDA receptors (Turesson and Sundin, 2003). Considering the AMPA dependent removal of the Mg2+ block to activate NMDA receptors (Monaghan et al., 1989), it is likely that both NMDA and AMPA receptors are involved in mediating the hypoxia activated sympathetic hypertension. However, mean arterial blood pressures during normoxic and hypoxic conditions are dependent on cardiac output and arterial resistances, why resistance changes both in the systemic and the branchial circulation needs to be monitored to fully resolve involvement of glutamatergic ionotropic receptors in hypoxia evoked resistance changes of the fish vasculature. 4.2.3. Kainate receptors and cardiovascular control The fish in the UBP296 treatment group did not show a pressor response to the NaCN injection. As the bradycardia was severe this most likely masks a potential increase in ventral aortic pressure. These results correspond to previous studies on responses to NaCN injections before and after MK801 treatment (Turesson and Sundin, 2003). Thus, chemoreflex mediation via NaCN induces a stronger bradycardia and an absent pressor response compared to when chemoreceptors are stimulated via hypoxic exposure. 4.2.4. Season-dependence of the role of non-NMDA receptors in control of heart rate That the summer control animals (GYKI52466 treatment group) in this study did not display a hypoxia sensitive bradycardia is in contrast with results from the winter animal in a previous study (Turesson and Sundin, 2003), as well as the spring animals from the UBP296 treatment group. Nevertheless, these results are in correspondence with previous studies on Atlantic cod where no bradycardia was detected during summer (Axelsson and Fritsche, 1991), compared to winter animals that did display bradycardia using the same experimental set-up and protocol (Fritsche and Nilsson, 1989, 1990). Moreover, ␤-adrenoceptor density is upregulated in cold-acclimated rainbow trout (Keen et al., 1993), which provides the fish with a higher sympathetic tonus on the heart. In turn, when a cold-acclimated fish is exposed to hypoxia the higher sympathetic tonus can then lead to a more apparent

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bradycardia. Thus, the different cardiac responses to hypoxia or NaCN injection may reflect season variation in receptor sensitivity or density in the summer animals in this study compared to the ones in the previous study by Turesson and Sundin (2003) and the spring animals. It is also plausible that the GYKI52466-induced rise in resting heart rate from the relatively lower values present in the fish before the blocker was administered, allowed for the hypoxic bradycardia to develop. 4.3. The role of non-NMDA receptors in breathing rhythm regulation Generally the aquatic branchial ventilation is rhythmic and regular, where arrhythmic and intermittent breathing develops with the transition to air breathing vertebrates (Smatresk, 1990). Indeed, many fish species like trout do breathe regularly but carp is an exception that displays irregular and intermittent breathing during normoxic conditions (Saunders, 1961; see Milsom, 1991). Shorthorn sculpin is another exception, where normal breathing rhythms in resting conditions can be quite irregular, since in normoxia most interbreath time intervals varied between 2 and 10 s, with occurring intervals as long as 60–65 s. Beside the influence of the afferent feed back on the respiratory performance to meet environmental and metabolic demands, regulation of the central rhythm/pattern generator activities may also shape the arrhythmic ventilatory behavior observed in shorthorn sculpins. Reflecting a putative influence of the latter is that normoxic untreated summer animals (GYKI52466 treatment group) and winter animals (MK801 treatment group) showed a season difference in the variability of the interbreath intervals in exactly the same experimental conditions, with the summer animals showing more than three times as high variability than that of the winter animals. Data exists that ionotropic receptors are important for producing breathing rhythms and shaping breathing patterns in several mammals including rats, cats, ground squirrels and rabbits (NMDA receptors, Connelly et al., 1992; Ling et al., 1994; Harris and Milsom, 2001; Mutolo et al., 2005, and non-NMDA receptors, Ge and Feldman, 1998; Whitney et al., 2000). Corroborating these findings are our results from shorthorn sculpins, where the NMDA receptor antagonist MK801 produces a more irregular breathing pattern while, GYKI52466 markedly produced a more regular breathing rhythm. Striking is that the breathing rhythms of MK801 treated winter animals are similar to untreated summer animals, while the appearance of GYKI52466 treated summer animals is similar to untreated winter animals. A theory for this relationship is that a down regulation of AMPA receptors occurs during winter, and down regulation of NMDA receptors occurs during summer, which then creates the observed variability in interbreath intervals between seasons. Indeed, there exist data that hypoxia downregulates and silences NMDA receptors to reduce hypoxic stress (Bickler et al., 2000; Kobayashi and Millhorn, 2000). Hypoxic conditions particularly occur in summer, and then especially in the benthic milieus of the shorthorn sculpin. The poincare’ plots for the UBP926 treatment group show much less variability in interbreath intervals. As the fish were kept slightly anesthetized to facilitate microinjections into the medulla this most likely affected the natural variability. 4.4. Distribution of non-NMDA receptors in the medulla The vagal sensory column earlier identified in the shorthorn sculpins (Sundin et al., 2003b) receives afferent information from the orobranchial cavity where the major part of the oxygen chemoreceptors is located (Hughes and Shelton, 1962; Saunders and Sutterlin, 1971; Sundin et al., 1999, 2000; Milsom et al., 2002). Data suggests that different oxygen receptor groups acti-

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vates different cardiorespiratory reflex modalities (Sundin et al., 1999, 2000; Milsom et al., 2002) and that each cardiorespiratory reflex appears to have a designated terminal field (sensory area) of their afferents in the medulla, since glutamate applied into different areas elicits different cardiovascular responses (Sundin et al., 2003a). To establish the location of non-NMDA receptors in medullary areas where glutamate elicits cardiorespiratory responses and AMPA and kainate receptors may participate in the control of the cardiorespiratory chemoreflexes, immunohistochemical analysis of AMPA and kainate receptor subunits in the whole vagal sensory area was performed. Dense labeling of GluR2/3 in nerve fibres and cell bodies throughout the vagal sensory column were found. In addition, both GluR2/3 and GluR5/6/7 were localized on vagal motor neurons. This is in accordance with the physiological experiments showing a role for non-NMDA receptors in evoking cardiorespiratory reflexes (present study) and a previous study where glutamate elicited cardiorespiratory responses when injected into different areas of the vagal sensory column (Sundin et al., 2003b). In addition, the immunohistochemical finding is in agreement with results obtained from rats, which show GluR2/3 and GluR5/6/7 subunits throughout the NTS (Kessler and Baude, 1999; Robinson and Ellenberger, 1997), on both fibers and neuronal cell bodies (Lacassagne and Kessler, 2000). Even though no GluR5/6/7-like IR was found in the vagal sensory area, there is still a possibility that the antibodies were not specific enough to stain fish kainate receptor subunit. Nevertheless, vagal motor neurons did get stained and this together with the fact that kainate receptors are involved in mediating the ventilation amplitude response opens up the possibility that there are first order glutamatergic afferents synapsing directly on vagal motor neurons. 4.5. Conclusions In conclusion, this study shows that AMPA receptors are involved in processing information eliciting oxygen chemoreflexes and regulating normoxic respiratory and cardiac rhythms. Furthermore, we have also shown that kainate receptors mediate the hypoxia-induced increase in ventilatory amplitude in fish. Finally, oxygen chemoreflex-activation in fish appears to be very complex. It involves several peripheral oxygen receptor groups with afferents projecting to different termination sites, where different glutamate receptor types mediate different cardiorespiratory responses. Acknowledgements This work was financially supported by grants from the Swedish Research Council (VR) and Helge Ax:son Johnsons Stiftelse. We are indebted to Jonas Eriksson, Lisette Fritzon, Emma Nohrén and Josefin Ragge for help with immunohistochemical experiments. References Ambalavanar, R., Ludlow, C.L., Wenthold, R.J., Tanaka, Y., Damirjian, M., Petralia, R.S., 1998. Glutamate receptor subunits in the nucleus of the tractus solitarius and other regions of the medulla oblongata in the cat. J. Comp. Neurol. 402, 75–92. Andresen, M.C., Yang, M.Y., 1990. Non-NMDA receptors mediate sensory afferent synaptic transmission in medial nucleus tractus solitarius. Am. J. Physiol. 259, H1307–H1311. Axelsson, M., Fritsche, R., 1991. Effects of exercise, hypoxia and feeding on the gastrointestinal blood flow in the Atlantic cod, Gadus morhua. J. Exp. Biol. 158, 181–198. Axelsson, M., Fritsche, R., 1994. Cannulation techniques. Biochem. Mol. Biol. Fishes 3, 17–36. Bickler, P.E., Donohoe, P.H., Buck, L.T., 2000. Hypoxia-induced silencing of NMDA receptors in turtle neurons. J. Neurosci. 20, 3522–3528. Bissonnette, J.M., Hohimer, A.R., Knopp, S.J., 1997. Non-NMDA receptors modulate respiratory drive in fetal sheep. J. Physiol. 501 (Pt 2), 415–423. Bongianni, F., Deliagina, T.G., Grillner, S., 1999. Role of glutamate receptor subtypes in the lamprey respiratory network. Brain Res. 826, 298–302.

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