Hypoxia attenuates the respiratory response to injection of substance P into the nucleus of the solitary tract of the rat

Neuroscience Letters 256 (1998) 9–12

Hypoxia attenuates the respiratory response to injection of substance P into the nucleus of the solitary tract of the rat Stuart B. Mazzone a, Colin F. Hinrichsen b, Dominic P. Geraghty a ,* a

School of Biomedical Science, University of Tasmania, Launceston, Tasmania 7250, Australia Discipline of Anatomy and Physiology, University of Tasmania at Hobart, Tasmania 7001, Australia

b

Received 20 July 1998; received in revised form 17 August 1998; accepted 1 September 1998

Abstract Prolonged or repetitive bouts of hypoxia may desensitize the brain stem respiratory centres leading to reduced stimulation of ventilation. We investigated the possible involvement of changes in the sensitivity of the commissural nucleus of the solitary tract (cNTS) to the tachykinin peptide, substance P (SP). Urethane-anaesthetised rats were allowed to breath room air (normoxic) or subjected to four, 30 s bouts of hypoxia (10% O2/90% N2) prior to the injection of SP (750 pmol) into the cNTS. In normoxic rats (n = 5), SP produced a fall in frequency (f, 88 ± 4% control) after 4 min and a maximum rise in tidal volume (VT) after 6 min (138 ± 10% control) leading to an overall increase in minute ventilation (VE, maximum, 127 ± 12% control after 2 min). In rats (n = 5) exposed to four bouts of hypoxia and allowed to recover for 10 min, injection of SP produced a similar fall in f but a delayed and significantly (P , 0.001) reduced VT (maximum after 10 min, 110 ± 1% control) and hence, VE response (104 ± 3% control). Sixty min after hypoxia, the f, VT and VE responses to SP were identical to those of normoxic rats. These data suggest that hypoxia desensitizes SP receptors in the cNTS and this may partly explain why the respiratory response to hypoxia declines over time.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Substance P; Hypoxia; Nucleus of the solitary tract; Respiration

The tachykinin substance P (SP) appears to be a key neurotransmitter of the peripheral chemoreceptor reflex. SP-containing afferent projections from carotid body chemoreceptors travel in the carotid sinus nerve and terminate in a number of regions of the nucleus of the solitary tract (NTS), including the commissural subnucleus (cNTS), and nearby dorsal motor nucleus of the vagus nerve [2,4,6,10,17]. Moreover, SP levels in the NTS are substantially elevated following hypoxic activation of the carotid body chemoreceptors [1,11]. Pharmacological studies also support a role for SP in chemoreceptor integration. Intracerebroventricular (i.c.v.), and microinjection of SP into various brain stem sites, stimulates ventilation, while injection of SP receptor (neurokinin-1; NK1) antagonists block these facilitatory effects [3,5].

* Corresponding author. P.O. Box 1214. Tel.: +61 3 63243379; fax: +61 3 63243658; e-mail: [email protected]

The mammalian respiratory response to hypoxia (RRH) is distinctly biphasic [7,12]. Both neonates and adults initially respond to hypoxia by hyperventilating (an increase in minute ventilation). However, if hypoxia is prolonged, hyperventilation gradually declines in the adult and may actually revert to hypoventilation in the neonate. The mechanisms underlying the biphasic RRH are not fully understood although failure to maintain hyperventilation during hypoxia has been linked to sudden infant death syndrome (SIDS). We have previously demonstrated that both single and multiple acute (5 min) bouts of hypoxia (8.5% O2 in N2) deplete the number of SP (NK1) receptors in key brain stem nuclei (including the NTS) involved in chemoreceptor reflex control of ventilation [15]. In the NTS, receptor depletion appears to be dynamic, reaching a maximum 5 min after hypoxia but returning to prehypoxic levels within 60 min. Presumably, this reduction in receptor numbers would attenuate the hyperventilatory effects of SP. Several

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00743- 5

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S.B. Mazzone et al. / Neuroscience Letters 256 (1998) 9–12

other laboratories have provided evidence for rapid, SPinduced desensitization and internalization of NK1 receptors in vitro [8,13]. However, to date there is little information regarding in vivo functional desensitization of this neurotransmitter system in the brain stem. Thus, the aim of the present study was to determine whether the temporary depletion of NK1 receptors which we have previously described in the NTS following hypoxia results in functional desensitization of the NTS to SP. All experimental procedures were approved by the University of Tasmania Ethics Committee (project 97044). Male Hooded Wistar rats (230–270 g) were anaesthetized using urethane (ethyl carbamate, 1–1.5 g/kg i.p. in two boluses, 15–20 min apart; Sigma, Melbourne, Australia). We and other research groups have demonstrated that hypoxia-induced hyperventilation is well maintained in urethane-anaesthetized rats [14,15,18]. The level of anaesthesia was assessed by monitoring limb withdrawal and head shake reactions. Body core temperature, measured using a thermistor inserted approximately 6 cm into the colon, was kept constant at 37°C by placing the rat in the prone position on a thermostatically controlled water bed. The animal’s head was stabilized in a Kopf stereotaxic apparatus in the horizontal position (i.e. the neck was flexed 3 mm from zero; Paxinos and Watson [16]), and the skull exposed by a midline incision. The dorsal aspect of the brain stem and cerebellum was exposed by a partial occipital craniotomy. The dura mater was temporarily left intact. Animals were allowed to stabilise for 20 min before continuing. For hypoxia experiments, the muzzle of the rat was inserted into a recess in the side of a piece of rubber tubing (2.5 cm, i.d.) through which a stream of air was delivered. Normoxic control rats (n = 5) were allowed to breath room air continuously. Hypoxia was induced in 10 rats by replacing the air stream with a 10% O2/90% N2 gas mixture. Rats were exposed to 4 × 30 s bouts of hypoxia and allowed to recover for 30 s between bouts. Respiration was recorded using subcutaneous electrodes and a calibrated impedance converter (UFI; Morro Bay, CA, USA) as previously described by our laboratory [14,15]. Animals which were exposed to hypoxia were returned to room air and randomly divided into two groups of five rats which were allowed to recover for either 10 or 60 min. Respiratory movements were displayed on the chart monitor and converted to frequency (f), tidal volume (VT) and minute ventilation (VE). To minimise inter-individual variation in respiratory activity (refer Table 1), the data for each animal was expressed as a percentage of its own control (basal or pre-SP injection) level. Data are presented as the mean ± SEM, and significant differences between group means were assessed using one-way analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference (LSD) test. P , 0.01 was considered statistically significant. Four bouts of hypoxia increased f, VT and VE but all respiratory indices had returned to control (normoxic)

values within 10 min of the fourth hypoxic challenge (Table 1). Interestingly, there was a progressive (but not statistically significant) decline in the maximum VT and VE responses to each successive bout of hypoxia: maximum VE (% control), 131 ± 4, 130 ± 7, 125 ± 7 and 119 ± 4; n = 10), suggestive of respiratory centre desensitisation. Following the hypoxia or normoxia protocol, the dura mater was cut and retracted. A microsyringe (o.d., 0.47 mm; Hamilton, Reno, Nevada) was mounted in a micromanipulator and using obex as a reference point, the needle was inserted (0.4 mm ventral) into the cNTS (midline, 0.5–0.8 mm caudal to obex; Paxinos and Watson [16]). SP (750 pmol in 1 ml normal saline; Auspep, Melbourne, Australia) was then slowly injected over 30 s. Respiratory movements were recorded continuously. At the end of each procedure, all animals were killed using an overdose of pentobarbitone sodium (Nebutal; Bomac, Australia). The brain stems were removed and fixed in formalin and stored frozen at −70°C. The injection sites were later located in cryostat cut sections. The needle tract was evident in the cNTS of all rats used in the study. In agreement with previous studies [9], microinjection of vehicle (1 ml of normal saline) into the cNTS had no effect on respiration (Fig. 1A). Injection of SP into the cNTS of normoxic rats produced a small decrease in f (88 ± 4% control) which returned to the pre-injection value by 20 min (Fig. 1B). In contrast, VT increased over the first 10 min (maximum, 138 ± 10% control at 6 min; Fig. 1A) and slowly returned to pre-injection levels by 60 min. Overall, VE increased for 10 min after the injection (maximum after 2 min, 127 ± 12% control) and gradually returned to normal by 40 min. In rats subjected to hypoxia and allowed to recover for 10 min, injection of SP produced a similar fall in f. However, the increase in VT (maximum after 10 min, 110 ± 1% control) was both delayed and significantly attenuated (P , 0.001; Fig. 1A,B). The attenuated VT response combined with reduced f led to a reduction in maximum VE response (104 ± 6% control after 10 min). Indeed, the present data suggest that injection of SP 10 min after hypoxia actually decreased VE below pre-injection (control) values. Table 1 Respiratory frequency, tidal volume and minute ventilation before and after hypoxia Treatment

f (breaths/min)

VT (ml)

VE (ml/min)

Normoxia Hypoxia (10 min recovery) Hypoxia (60 min recovery)

124 ± 8 115 ± 3

2.3 ± 0.2 2.3 ± 0.1

290 ± 33 268 ± 15

111 ± 5

2.2 ± 0.1

244 ± 6

Respiratory frequency (f), tidal volume (VT) and minute ventilation (VE) were measured in normoxic rats and in rats which were allowed to recover for 10 and 60 min from four bouts of hypoxia. Data are the mean ± SEM of five rats.

S.B. Mazzone et al. / Neuroscience Letters 256 (1998) 9–12

Fig. 1. (A) Chart tracings which show respiratory movements of normoxic rats and hypoxic rats 30 s before and 6 min after injection of substance P (SP; 750 pmol) into the commissural nucleus of the solitary tract (cNTS). The uppermost traces show the effect of vehicle (normal saline). (B) Mean respiratory frequency (f), tidal volume (VT) and minute ventilation (VE) following the injection of SP into the cNTS of normoxic rats (-X-) and rats allowed to recover for 10 min (O-) or 60 min (-W-) from four bouts of hypoxia. Data are expressed as a percentage of individual normoxic (or pre-hypoxic) value and shown as the mean ± SEM of five rats. †P , 0.01; *P , 0.001, significantly different from normoxic rats.

However, the decrease in overall ventilation was not statistically different from pre-injection values. When rats were allowed to recover for 60 min from hypoxia, the f, VT and VE responses to injection of SP were almost identical to those of the normoxic group. Since a relatively large injection volume was used in the present study, we also investigated the diffusion of SP away from the injection site. SP (750 pmol in 1 ml), supplemented with approximately 5 fmol (50 000 dpm) of the NK1 selective radioligand, [125I]Bolton Hunter SP (BHSP; 2200 Ci/ mmol; New England Nuclear, Boston, MA, USA) was injected into the cNTS. Animals were allowed to survive for 10 min before the brain was rapidly removed and frozen. Cryostat-cut horizontal sections (10 mm) of brain stem were thaw-mounted onto gelatin-coated slides and desiccated overnight at 4°C. Slides were then fixed in paraformaldehyde vapor (2 h at 80°C) and apposed to 3H-sensitive Hyperfilm for 4 days along with [125I]Microscale standards (Amersham, Buckinghamshire, UK). Hyperfilms were developed under safe-light conditions and gray scale densities converted to dpm/mm2 using image analysis (AIS; Analytical Imaging Station, Imaging Research, St. Catherines, Ontario). To determine lateral spread, a 65 mm sampling

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tool was used to measure the density adjacent to the needle tract (which generally displayed the highest density) of sections which showed the greatest degree of spread. Subsequently, sections were sampled at 125 mm intervals. To determine ventral and dorsal spread, the section which displayed the highest density of BHSP was sampled and used as the reference point. For each section ventral and dorsal to the reference point, the area of highest density was sampled and expressed as a percentage of the reference value. Quantitation of autoradiograms (Fig. 2A) showed that the concentration of BHSP declined by approximately 50 percent at a distance of 0.8 mm lateral to the injection site and by a further 23% at 1.2 mm (Fig. 2B). Lateral spread of BHSP beyond 1.3 mm of the injection site was negligible. Ventral and dorsal spread were more confined. The maximum concentration of BHSP declined by approximately 80% ventral and 50% dorsal at a distance of 0.36 mm of the injection site (Fig. 2C). Although BHSP and SP would obviously have slightly different diffusion rates due to differences in molecular weight and size, the BHSP/SP coinjection experiment gives some indication as to the spread of SP from the injection site. Based on our calculations, we predict that the actions of injected SP would be limited to the caudal NTS, dorsal motor nucleus of the vagus and nearby area postrema. Although the area postrema possesses a high density of NK1 receptors [15], a respiratory role for tachykinins in the area postrema of the rat has not been reported in the literature. The present data confirm previous studies which show that injection of SP (i.c.v. or localised into the brain stem) increases VT [3,9]. Our previous findings showed that a single hypoxic episode or multiple bouts of hypoxia leads to a temporary depletion of NK1 receptors in several brain stem regions associated with respiratory control, including the NTS [15]. The maximum depletion of NK1 receptors in the NTS occurs 5 min after a single bout of hypoxia, while normal receptor complement is restored after 60 min. In the present functional studies, the respiratory (depth) response to microinjection of SP into the cNTS was markedly attenuated 10 min after hypoxia but was identical to that of normoxic rats after 60 min. Thus, one might suggest that the attenuation and subsequent restoration of the respiratory response to SP after hypoxia are related, at least in part, to the loss and replenishment respectively of NK1 receptors in the NTS. In conclusion, the present study suggests that brief bouts of hypoxia, which causes the release of SP from the central terminals of carotid body afferents, markedly desensitizes the NTS to the excitatory actions of SP. This phenomenon may be related to agonist-induced internalization of NK1 receptors at this key chemoreceptor integration site. A reduction in the excitatory effects of SP on the respiratory centres of the brain stem may partly explain why adequate ventilation is not always maintained during hypoxia, even with continued carotid body input. Since failure to maintain hyperventilation during hypoxia may underlie the pathogen-

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Fig. 2. (A) Scanned positives of [3H]Hyperfilm images which show the amount of [125I]Bolton-Hunter substance P (BHSP) present in horizontal sections (10 mm, 50 mm between sections) of brain stem after injection into the commissural nucleus of the solitary tract. Numbers refer to ventral (V) and dorsal (D) distance from the injection point (0). (B) Lateral spread of BHSP. The amount of BHSP (expressed as percent maximum observed) was measured every 125 mm lateral to the injection point (initial measurement at 65 mm from center of needle tract) on sections which showed the maximum degree of spread. (C) Ventral and dorsal spread of BHSP after injection into the cNTS. Each point in (B) and (C) represents the mean ± SEM of three rats.

esis of SIDS, we propose that a dysfunction of SP neurotransmission may be a contributing factor. This study was supported by the National Health and Medical Research Council of Australia (DPG) and the National SIDS Council of Australia (DPG, CFH). The authors wish to thank Mrs. Leonie Agius for assistance in preparing figures.

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