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Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity
Respiratory Physiology & Neurobiology 138 (2003) 165 /178 www.elsevier.com/locate/resphysiol
Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity Tina Lonergan a,b,1, Ann K. Goodchild b, Macdonald J. Christie a, Paul M. Pilowsky a,b,* b
a Department of Pharmacology, University of Sydney, Camperdown, NSW 2006, Australia Hypertension and Stroke Research Laboratories, Department of Physiology, University of Sydney and Department of Neurosurgery, Royal North Shore Hospital, Ground Floor, Block 3, St. Leonards, NSW 2065, Australia
Accepted 4 June 2003
Abstract Anatomical and in vitro studies suggest that mu opioid receptors (MOR) on pre-Bo¨tzinger complex neurons are responsible for opioid induced respiratory depression (Grey et al., Science 286 (1999) 1566). However, mu opioid agonists injected in vivo, in other regions of the ventral respiratory group (VRG), produce respiratory depression, suggesting that opioids are widely distributed in the VRG. We therefore re-examined the distribution of the MOR in the ventral medulla and found MOR-immunoreactive neurons and terminals in all subdivisions of the VRG. Furthermore, we determined, in rats, the effects of a MOR agonist (endomorphin-1, 10 mM, 60 nl, unilateral), microinjected into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 produced changes in phrenic nerve frequency and amplitude, throughout the VRG. Unexpectedly, endomorphin-1 microinjected into the Bo¨tzinger and pre-Bo¨tzinger complexes consistently increased phrenic nerve frequency. These results support the widespread distribution of MOR in the VRG and also indicate that endomorphin-1, a postulated endogenous ligand, may differentially regulate respiration. # 2003 Elsevier B.V. All rights reserved. Keywords: Brainstem; Pre-Bo¨tzinger region; Mu opioid receptors; Control of breathing; Respiratory depression; Mammals; Rat; Receptors; Mu opioid
1. Introduction The mu opioid receptor (MOR) is implicated in the respiratory depressant actions of opioids in the medulla. Early, in vivo, work demonstrated that
intracerebroventricular (Pazos and Florez, 1983; Hurle et al., 1985), or ventral medullary surface application (Hurle et al., 1985) of MOR selective agonists caused respiratory depression, however, the exact neuronal structures involved in MOR
* Corresponding author. Tel.: /61-2-9926-8080; fax: /61-2-9926-6483. http://www.physiol.usyd.edu.au/ /pilowsky/. E-mail address: [email protected] (P.M. Pilowsky). 1 Formerly Stasinopoulos. 1569-9048/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1569-9048(03)00173-3
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induced respiratory depression remain loosely defined. Gray et al. (1999) suggested that opioid respiratory depression is due to inhibition of rhythm generating neurons in the pre-Bo¨tzinger complex and that MOR-immunoreactivity (ir) is exclusively found in that area. While the involvement of the pre-Bo¨tzinger complex in MOR induced respiratory depression is well established in neonatal in vitro preparations (Murakoshi et al., 1985; Greer et al., 1995; Johnson et al., 1996; Ballanyi et al., 1997; Takita et al., 1997; Gray et al., 1999), other structures may be involved in the adult, in vivo. In adult rats and cats, iontophoresis of opioids onto respiratory units throughout the ventral respiratory group (VRG) inhibited their activity (Denavit-Saubie´ et al., 1978; Rondouin et al., 1981) and microinjection of the selective MOR agonist, DAMGO, into the rostral VRG (rVRG) or the nucleus ambiguus (NA), depressed respiration (Hassen et al., 1984; Chen et al., 1996). These data suggest that the MOR is widely distributed in the VRG of the adult rat, and that it modulates the activity of a wide range of respiratory neurons. Therefore the aims of this study were, firstly, to re-examine the distribution of the MOR in the ventral medulla. Secondly, to determine the effects of microinjection of endomorphin1, into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 was chosen as it is suggested to be an endogenous ligand for the MOR (Zadina et al., 1997). Furthermore, unlike other endogenous peptides, which are only 10 / more selective for the MOR over the delta opioid receptor (DOR) (Goldstein and Naidu, 1989), endomorphin-1 is more than 4000 / selective for the MOR over the DOR (Zadina et al., 1997).
approved by the Animal Care and Ethics Committee of the Royal North Shore Hospital. 2.1. Immunohistochemistry Rats (n/3) were anaesthetised (sodium pentobarbital, 100 mg/kg, i.p.) and intracardially perfused with DMEM (Sigma), followed by 4% formaldehyde in 0.1 M phosphate buffer (PB), at pH 7.4. Brainstems were removed and post-fixed for 1.5 h and serial 50 mm sections were cut using a vibrating microtome. The sections were washed for 30 min in 50% ethanol, followed by 3/30 min washes in tris-phosphate buffered saline (TPBS; Tris /HCl 10 mM, sodium phosphate buffer 10 mM, 0.9% NaCl, pH 7.4). Sections were incubated in a rabbit anti-MOR1 antibody (1:20 000, Incstar) and 5% normal horse serum (NHS) in TPBS, for 72 h. This was followed by overnight incubations in biotinylated-donkey anti rabbit F(ab?)2 fragments (1: 500, Jackson: 711-066-152) and 2% NHS in TPBS, then ExtrAvidin-HRP in TPBS (1:1000, Sigma). MOR-ir was revealed using a nickel intensified, glucose-oxidase peroxidase reaction (Llewellyn-Smith et al., 1992). Inclusion of the peptide (10 mg/ml) against which the antibody was raised, abolished MOR-ir, suggesting the antibody was specific. Washes were performed between incubations (TPBS, 3 /30 min), at room temperature. Sections were serially mounted, dehydrated, and coverslipped using Ultramount (Fronine Pty Ltd, Australia). Sections were viewed using a Leica DML microscope and images were obtained using a Spot2 digital camera (Diagnostic Instruments), contrast and brightness adjusted for using Spot2 proprietary software. 2.2. Microinjections
2. Experimental procedures Adult male Sprague/Dawley rats (200 /500 g) were used. Experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes as endorsed by the National Health & Medical Research Council of Australia. Protocols were
Rats (n/8) were anaesthetised with urethane (1.3 g/kg, i.p.), additional doses given as required (0.2 g/kg, i.v.). Atropine sulphate (0.15 mg/kg, i.p.) was administered to reduce airway secretions. The trachea, left femoral artery and vein were cannulated. The cervical vagus, phrenic nerve and the facial nerve were exposed. Rats were placed in a stereotaxic frame, ventilated, paralysed, bilaterally
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vagotomised and the dorsal surface of the medulla exposed. End tidal CO2 was monitored and maintained between 4 and 5%. The vagus and phrenic nerves were placed on bipolar silver electrodes for stimulation and recording, respectively. Bipolar stimulating electrodes were hooked onto the facial nerve. Phrenic nerve discharge was acquired at a gain of 20 000 /50 000 (AC-coupled preamplifier and amplifier, CWE), band pass filtered (10 /1 kHz), digitised and sampled at 2000 Hz (1401-plus, CED, UK). Multibarrel glass pipettes were constructed and filled with 3 M NaCl, endomorphin-1 (10 mM, in 0.01 M PBS, pH 7.4, Chiron Technologies, Australia) and albumin/colloidal gold (20% in 0.01 M PBS, pH 7.4, Sigma). The location of the facial nucleus (VIIn), compact formation of the nucleus ambiguus (NAc) and respiratory field activity were mapped by antidromic stimulation of the facial and vagus nerve, as previously described (Brown and Guyenet, 1985; Pilowsky et al., 1990; Sun et al., 1998). The extracellular signal was acquired with a 10 / gain (Axoclamp 2B, Axon Instruments), further amplified (200 /1000/) and band pass filtered (30 /3 kHz, AC Bioamplifier, CWE), digitised and sampled at 2000 Hz (CED 1401plus). Unilateral microinjections of endomorphin-1 (60 nl) were made in regions where respiratory field activity could be recorded. The effect on phrenic nerve discharge and mean arterial pressure (MAP) was monitored for at least 30 min, following each injection. Injections of colloidal gold (60 nl) were made following each endmorphin-1 injection. At the end of the experiment, the rat was euthanised (3 M KCl, i.v.) and the brainstem removed and fixed overnight. Sections (100 mm) were cut using a vibrating microtome and processed for gold injection sites using a silver enhancer kit (Sigma). Sections were mounted on gelatinised slides, dehydrated, counterstained using cresyl violet, and coverslipped.
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2.3. Data analysis 2.3.1. Analysis of phrenic nerve discharge Phrenic nerve activity was analysed offline using SPIKE2 software (CED). Raw phrenic nerve activity was rectified and integrated as a moving average with a 20 msec time constant. Waveform averages and mean frequencies of phrenic nerve discharge were obtained over 2 min periods. The inspiratory time, expiratory time, respiratory cycle time and peak amplitude of phrenic nerve discharge were calculated from waveform averages. Pre-drug values were compared to post-drug values at the following times: 1, 5, 10, 15, 20, 25 and 30 min post-injection. For most of the injection sites, phrenic nerve activity was no longer significantly different from control at 30 min (significance was taken at /15% difference from control). When the effects were still significant at 30 min, measurements were taken approximately every 10 min until the changes were no longer significantly different to control. Apnoea was defined as the absence of phrenic nerve discharge for periods greater than 3 sec. 2.3.2. Determination of location of injection sites The centre of injection sites was determined and plotted onto brainstem maps (Swanson, 1998) with reference to the caudal border of the VIIn (bregma /12.75 mm).
3. Results 3.1. Immunohistochemistry Intense MOR-ir was observed in many regions of the medulla, including the area postrema, dorsal motonucleus of the vagus, solitary tract and nucleus, spinal trigeminal nucleus, inferior olive, and the NAc, as previously reported (Arvidsson et al., 1995; Ding et al., 1996). Less intense MOR-ir was present throughout the medulla, including the VRG (Fig. 1). Small numbers of weakly MOR-ir neurons were found throughout the rostrocaudal extent of the VRG (Fig. 1), while MOR-ir fibres and terminals were more abundant (Fig. 1). There appeared to be no difference in the number of
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Fig. 1. Distribution of MOR-ir in the region of the VRG. (A) cVRG, (B) rVRG, (C) pre-Bo¨tzinger complex, (D) Bo¨tzinger complex. The boxed areas in the low power images are shown in the high power images on the right. In the region of the VRG, numerous MORir neurons(*), fibres (arrow) and varicosities (arrow heads) were observed. Intense MOR-ir was observed in the area postrema, dorsal motonucleus of the vagus, solitary tract and nucleus, spinal trigeminal nucleus, inferior olive, the NAc and vagal afferents in the rostral medulla. Scale bars are 100 mm.
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MOR-ir neurons in the region of the pre-Bo¨tzinger complex compared to the adjacent Bo¨tzinger complex and rVRG. A group of intensely labelled MOR-ir neurons and fibres were observed dorsomedial to where the pre-Bo¨tzinger is thought to lie (Fig. 1C). The rostrocaudal extent of these neurons overlaps with that of the pre-Bo¨tzinger, although these neurons appear to extend rostrally for another 200 mm. 3.2. Microinjection of endomorphin-1 into the VRG Endomorphin-1 was microinjected into 23 sites in the ventral medulla, spanning the Bo¨tzinger complex, pre-Bo¨tzinger complex and rVRG (defined by rostrocaudal location and respiratory field activity at the site of injection (Sun et al., 1998). Figs. 2/4 show the injection sites and effects of endomorphin-1 on phrenic nerve discharge frequency (Fig. 2), amplitude (Fig. 3) and MAP (Fig. 4). Injection of albumin-colloidal gold suspended in the same vehicle as endomorphin-1, did not alter phrenic nerve activity or MAP, confirming that the effects seen were due to the actions of endomorphin-1. 3.2.1. Bo ¨ tzinger complex A total of six injections of endomorphin-1 were made into the Bo¨tzinger complex, in sites of expiratory (5) or E-I field activity (1), 100/700 mm caudal to the caudal border of the VIIn (Fig. 2i, Fig. 5). Endomorphin-1 injected into the Bo¨tzinger complex increased phrenic nerve frequency by 16 /30%, within the first 10 min, at 4/6 sites, (Fig. 2i, Fig. 5A). The responses usually disappeared within 20 min (Fig. 2i). At one site, endomorphin-1 elicited a decrease in phrenic nerve frequency that was followed by apnoea (Fig. 5B). The changes in frequency occurred as a result of shortening (Fig. 5A) or lengthening (Fig. 5B) of expiratory period. Endomorphin-1 attenuated phrenic nerve amplitude by 16/100%, at 5/8 sites (Fig. 3i). At 2 of these sites, apnoea occurred (e.g. Fig. 5B). At one site, endomorphin-1 produced an increase in amplitude, with a maximal increase of 37% at 20 min post-injection. MAP was altered by
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endomorphin-1 at 2/6 sites in the Bo¨tzinger complex (Fig. 4i), injection at one site producing a depressor response, the other site producing a pressor response. 3.2.2. Pre-Bo ¨ tzinger complex A total of 10 microinjections of endomorphin-1 were made into the pre-Bo¨tzinger complex, in sites of inspiratory (6), expiratory (3) or E-I field activity (1), 0.75 /1.25 mm caudal from the caudal border of the VIIn (Fig. 2ii, iii, Fig. 3ii, iii, Fig. 4ii, iii). A reduction in frequency was only observed at 1/10 injection sites in the pre-Bo¨tzinger complex. At 8/10 sites, endomorphin-1 increased the frequency of phrenic nerve discharge by 16 /45% (Fig. 2ii, iii). The increase in frequency usually lasted for about 10 min (5/8 sites), but in some cases (3/8) lasted for 20/30 min (e.g. Fig. 6A). Changes in frequency occurred as a result of changes in both the inspiratory and expiratory period (e.g. Fig. 6A). Injection of endomorphin-1 at 7/10 sites reduced phrenic nerve amplitude by 16/50% (e.g. Fig. 6A) and the response lasted anywhere between 10 /30 min, (Fig. 3ii, iii). Injection of endomorphin-1 at 3/10 sites increased phrenic nerve amplitude by 16/30%. Excitatory and inhibitory effects on amplitude could not be distinguished by location or type of respiratory field activity at the site of injection. Endomorphin1 evoked depressor responses at 4/10 sites (Fig. 4ii, iii). 3.2.3. rVRG A total of seven injections of endomorphin-1 were made into the rVRG, in sites of inspiratory field activity, 1.30 /2.05 mm caudal from the caudal border of the VIIn (Fig. 2iv, v). Endomorphin-1 only produced effects on frequency at 3/7 sites in the rVRG. Injection of endomorphin-1 at one site increased frequency by 32% in the first 10 min. Endomorphin-1 injected at 2 sites produced a decrease in frequency, lasting 10/20 min, apnoea occurring at one of these sites (Fig. 6B). The changes in frequency caused by endomorphin-1 were largely due to changes in expiratory period. Endomorphin-1 produced a decrease in phrenic nerve amplitude at 6/7 sites (e.g. Fig. 6B). This fall in amplitude lasted between
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Fig. 2. Time course of effects (left to right) of unilateral microinjections of endomorphin-1 (10 mM, 60 nl), into different rostrocaudal levels of the VRG on phrenic nerve discharge frequency. Each circle or square represents an injection site and the increasing darkness and size relates to increasing changes that occurred during that particular time period.
10 /60 min (Fig. 3iv, v) and elicited a depressor response at 2/7 sites (Fig. 4iv).
4. Discussion This study demonstrates MOR-ir on neuronal perikarya and terminals throughout the rostrocaudal extent of the VRG. Supporting the widespread distribution of MOR-ir in the VRG, endomorphin-1 produced changes in both phrenic nerve amplitude and frequency in all regions of the VRG tested.
4.1. Relationship between MOR-ir and subdivisions of the VRG A small number of weakly MOR-ir neurons were observed throughout the ventral respiratory column. The number of MOR-ir neurons observed was far less than the number of NK-1-ir neurons that have been reported in the same region (Wang et al., 2001). The low level of MOR-ir in the VRG suggests that either the respiratory effects of opioids are mediated by a small number of receptors or that the MOR antibody is not sensitive enough to detect all the receptors in the area.
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Fig. 3. Same data as in Fig. 2, but plotting changes in phrenic nerve amplitude.
Gray et al. (1999) proposed that MOR-ir was confined to the pre-Bo¨tzinger complex and therefore could be used as neurochemical/anatomical marker of the pre-Bo¨tzinger complex (Gray et al., 1999). However, in this study MOR-ir was also found in the adjacent Bo¨tzinger complex, rVRG and cVRG. The reason for the discrepancy between these studies is not clear. While the same antibody was used in both studies, female rats and mice were used in Gray’s study, and the exact age was not specified. This may bear on the results since it was shown that MOR-ir decreases in the pre-Bo¨tzinger with age (O’Neal et al., 2000). Furthermore, thin cryostat sections and tyramine signal amplification was used to amplify MOR-ir in some of the experiments of Gray et al. (1999).
The differences in processing may explain the enhanced detection of MOR-ir in the pre-Bo¨tzinger; however, they do not explain why comparable MOR-ir was not detected in the adjacent Bo¨tzinger and rVRG by Gray et al. (1999). The pharmacological findings that MOR activation in the Bo¨tzinger and rVRG produced changes in phrenic nerve activity supports the immunohistochemical evidence presented in this study. The widespread distribution of MOR-ir neurons and terminals in the ventral medulla suggests that opioids, both endogenous and exogenous may directly inhibit, or inhibit afferent inputs to, respiratory neurons throughout the VRG. However, the usual physiological function of the MORir structures detected is unknown and they may
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Fig. 4. Same data as in Fig. 2, but plotting changes in MAP.
subserve respiratory, cardiovascular, or other functions. For example, C1 and sympathoexcitatory neurons in the rostral ventrolateral medulla are MOR-ir or receive synaptic inputs that are MOR-ir (Aicher et al., 2001a,b; Milner et al., 2001). The present study defined for the first time, a group of intensely stained MOR-ir neurons that were located dorsomedially to the MOR-ir neurons defined by Gray et al. (1999). The function of these MOR-ir neurons is unknown. Interestingly, the region where these MOR-ir neurons are located corresponds well with the gasping centre described by St.-John and colleagues in the rat (Fung et al., 1994, 1997; St.-Jaques and St.-John, 1999). The region also overlaps with sites that produced apnoea when DAMGO (a MOR ago-
nist) was microinjected into the rat (Hassen et al., 1984). Together, these data suggest that the medial MOR-ir neurons may be a part of the respiratory network. 4.2. Effects of endomorphin-1 in the VRG 4.2.1. Technical considerations Caution must be taken in extrapolating these results to the intact, conscious animal since the use of anaesthesia and vagotomy may remove tonically active inputs that may be modulated by the activation of MOR. Furthermore, unilateral injections may produce different responses compared to bilateral microinjections, since the contralateral side of the VRG receives inputs from the ipsilateral side (Ezure, 1990; Jiang and Lipski, 1990;
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Fig. 5. Effects of endomorphin-1 (10 mM, 60 nl, unilateral) microinjected into two sites in the Bo¨tzinger complex. (A) Endomorphin-1 injected into this site of expiratory field activity, produced a small reduction in phrenic nerve amplitude and a 30% increase in phrenic nerve frequency during the first 10 min. The increase in frequency was due to a decrease in expiratory period (see waveform average). (B) Endomorphin-1 injected into this site reduced phrenic nerve amplitude and frequency, leading to apnoea. MAP was initially reduced and then became erratic and did not return to normal until phrenic nerve activity recovered. The apnoea was due to a prolongation in expiratory time (see waveform average).
Nu´n˜ez-Abades et al., 1991; Gaytan et al., 1997; Wang et al., 2001) and may act to restore phrenic nerve activity. No previous study has microinjected endomorphin-1 into the brainstem, so a dose of 10 mM was used. Activation of the DOR by endomorphin-1 at the dose used, cannot be ruled out in these experiments. It has also been suggested that endomorphin-1 is a partial agonist in some tissues (Connor et al., 1999; Horvath, 2000), however the partial-agonist activity of endomorphin-1 in the medulla is undefined. Antagonists were not used in these experiments since it was observed that
desensitisation occurred following repeated administration of endomorphin-1 in the same site.
4.2.2. Comparison with other studies Studies where opioids were applied to the ventral/dorsal brainstem surface, microinjected intracerebroventricularly or into the medulla, report similar time courses to this study, with the time to peak ranging from 1 to 30 min and duration of effect ranging from 30 min to more than 2 h (Pazos and Florez, 1983, 1984; MorinSurun et al., 1984; Hurle et al., 1985; Chen et al.,
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Fig. 6. Effects of endomorphin-1 (10 mM, 60 nl, unilateral) microinjected into a site of expiratory field activity in the pre-Bo¨tzinger complex (A) and a site of inspiratory field activity in the rVRG (B). Endomorphin-1 produced a large reduction of phrenic nerve amplitude at both sites but produced an increase (25%) of phrenic nerve activity in the pre-Bo¨tzinger complex, but a decrease in frequency, that led to apnoea, in the rVRG. At both sites, the changes in frequency were due to alteration of the expiratory period (see waveform averages). There was little change in MAP at these sites.
1996; Takita et al., 1997; Janczewski and Feldman, 2000). In vivo microinjection studies using selective MOR agonists in the VRG are rare. No single study has mapped the effects of MOR agonists in different subdivisions of VRG. Instead, they focused on particular areas. For example, DAMGO microinjected unilaterally into the region of the NAc in the rat produced a decrease in respiratory frequency, instead of the increase seen
with endomorphin-1 in this study (Hassen et al., 1984). An increase in respiratory frequency was observed in several studies following intravenous administration of specific MOR agonists in conscious animals (Negri et al., 1998; Czapla et al., 2000), however activation of MOR in other regions of the brain may have contributed. DAMGO in the rVRG of the rat produced a decrease in frequency and amplitude (Chen et al., 1996), similar to endomorphin-1 in this study.
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Surprisingly, in the present study, apnoea was never evoked following microinjection of endomorphin-1 into the pre-Bo¨tzinger complex. This is contrary to in vitro (Greer et al., 1995; Johnson et al., 1996; Ballanyi et al., 1997; Takita et al., 1997; Gray et al., 1999) and in vivo studies (Janczewski and Feldman, 2000) that attribute opioid respiratory depression to MOR-induced inhibition of rhythm generating neurons in the pre-Bo¨tzinger complex. The differences between the current data and in vitro studies may reflect maturational changes (Paton et al., 1994; Paton and Richter, 1995; Ptak and Hilaire, 1999; Zhang et al., 1999), the absence of afferent inputs in vitro and the presence of anaesthetic in vivo. The differences seen between other in vivo experiments and the present study could reflect the difference in the experimental model used, for example, bilateral vs. unilateral microinjections, different anaesthetics (pentobarbitone vs. urethane), vagotomised vs. vagus intact, artificially ventilated vs. spontaneously breathing, and finally, the use of different ligands. 4.2.3. Mechanism of action of endomorphin-1 in the VRG 4.2.3.1. Bo ¨ tzinger complex. The increase in phrenic nerve discharge frequency following endomorphin-1 injections into the Bo¨tzinger complex may have resulted from an inhibition of excitatory inputs to Bo¨tzinger neurons or hyperpolarisation of Bo¨tzinger neurons, such that expiration was terminated earlier. The depression of phrenic nerve amplitude following endomophin-1 was possibly due to removal of inhibitory inputs to Bo¨tzinger neurons resulting in increased inhibitory activity to pattern generating neurons, inspiratory premotoneurons or phrenic motoneurons (Ezure and Manabe, 1988; Ezure, 1990; Jiang and Lipski, 1990; Tian et al., 1998, 1999a,b). 4.2.3.2. Pre-Bo ¨ tzinger complex. The increase in phrenic nerve discharge frequency following microinjection of endomorphin-1 into the pre-Bo¨tzinger complex may be due to the removal of inhibitory inputs to the pre-Bo¨tzinger, e.g. from
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the Bo¨tzinger complex. Since the increase in frequency was due to changes in both expiratory and inspiratory timing, activation of the MOR by endomorphin-1 in the pre-Bo¨tzinger affects mechanisms that determine both the inspiratory on and off switches. This is in contrast to the Bo¨tzinger complex and rVRG, where the MOR largely affected the length of expiration and thus the inspiratory on-switch. The observation of mixed effects on phrenic nerve amplitude following endomorphin-1 microinjection into the pre-Bo¨tzinger complex suggests that the MOR is found on more than one type of neuron in the pre-Bo¨tzinger complex, or on more than one afferent input to the pre-Bo¨tzinger. This is in contrast to the theory proposed by Gray et al. (1999) that the MOR is only found on type-I pre-I neurons. Indeed the immunohistochemical studies described here show that MOR-ir is observed on both terminals and neurons throughout the VRG.
4.2.3.3. rVRG. In the rVRG endomorphin-1 usually reduced phrenic nerve frequency suggesting a differentiation in the function of the MOR (or endomorphin-1) in the modulation of rhythm generation in the rVRG compared to the Bo¨tzinger and pre-Bo¨tzinger complexes. The depression of both phrenic nerve amplitude and frequency in the rVRG indicates that the role of the MOR in the VRG is to suppress the excitatory function of the bulbospinal neurons (either by inhibiting excitatory inputs and/or by hypopolarising bulbospinal neurons). Indeed Lalley (personal communication) has shown that DAMGO hyperpolarises augmenting bulbospinal inspiratory neurons in vivo. The occurrence of apnoea at one site further supports this idea. In all the regions discussed above there is also a possibility that some of the effects produced on phrenic nerve activity were due to activation of opioid receptors on the distal dendrites of respiratory neurons located in other regions of the VRG. For example, effects observed in the Bo¨tzinger complex may be due to actions on the dendrites of pre-Bo¨tzinger neurons located in this area.
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4.2.4. Effect of endomorphin-1 on MAP MAP was not affected greatly by injections of endomorphin-1, despite the recent identification of pre- and post-synaptic MOR-ir on functionally identified presympathetic neurons (Aicher et al., 2001a,b; Milner et al., 2001). The lack of effect may be due to the partial agonist activity of endomorphin-1. Other studies in our lab have shown that DAMGO, a full MOR agonist caused a significant depressor response when injected in the rostral ventrolateral medulla (Miyawaki et al., 2002).
lead compound in the search for opiates with less respiratory depressant effects than current agents. The multitude of responses and the location of the MOR in more than one region of the VRG suggest that the MOR plays a diverse role in the VRG. The effect of activation of the MOR, on central respiratory output is likely to depend on the endogenous ligand released, the location of release and the afferent inputs and respiratory neurons affected.
Acknowledgements 4.3. Endogenous endomorphin The mRNA and precursor protein that produces endomorphin-1 is unidentified. Endomorphin-1-like-ir terminals are distributed throughout the rat brain, including the brainstem (Zadina et al., 1999). Endomorphin-1-like-ir cell bodies are found in the hypothalamus and nucleus tractus solitarius (NTS), (Zadina et al., 1999), with a distribution akin to that of b-endorphin. The NTS could be a source of endomorphin-1 inputs to the VRG, as it projects to the VRG (Ellenberger and Feldman, 1990; Nu´n˜ez-Abades et al., 1993). Endomorphin-1-like-ir does not appear at birth and only matures at 21 days (Barr and Zadina, 1999), indicating that the function and activity of the MOR may change as the respiratory network matures. 4.4. Conclusion We have shown that MOR-ir is present throughout the VRG. Furthermore, activation of the MOR, by microinjection of endomorphin-1, into the Bo¨tzinger, pre-Bo¨tzinger or rVRG results in profound changes in both phrenic nerve frequency (rhythm generation) and phrenic nerve amplitude (pattern formation). However, each subdivision of the VRG produced a unique response to endomorphin-1. Perhaps the most surprising finding was that endomorphin-1 increased phrenic nerve frequency and thus promoted rhythm generation in the Bo¨tzinger and pre-Bo¨tzinger complex. This suggests that endomorphin-1 may be an interesting
Our laboratory is supported by grants from the National Health and Medical Research Council (980077, 211023), National Heart Foundation (GOOS0716) and North Shore Heart Research Foundation. M.J. Christie is supported by the University of Sydney Medical Foundation. T. Lonergan is supported by an Australian Postgraduate Award.
References Aicher, S., Kraus, J.A., Sharma, S., Patel, A., Milner, T.A., 2001a. Selective distribution of mu-opioid receptors in C1 adrenergic neurons and their afferents. J. Comp. Neurol. 433, 23 /33. Aicher, S., Schreihoffer, A.M., Kraus, J.A., Sharma, S., Milner, T.A., Guyenet, P.G., 2001b. Mu-opioid receptors are present in functionally identified sympathoexcitatory neurons in the rat rostral ventrolateral medulla. J. Comp. Neurol. 433, 34 /47. Arvidsson, U., Reidl, M., Chakrabarti, S., Lee, J.-H., Nakano, A.-H., Dado, R.J., Loh, H.H., Law, P.-Y., Wessendorf, M.W., Elde, R., 1995. Distribution and targeting of a mu opioid receptor (MOR1) in brain and spinal cord. J. Neurosci. 15, 3328 /3341. Ballanyi, K., Lalley, P.M., Hoch, B., Richter, D.W., 1997. cAMP-dependent reversal of opioid and prostaglandin mediated depression of the isolated respiratory network in newborn rats. J. Physiol. (London) 504, 127 /134. Barr, G.A., Zadina, J.E., 1999. Maturation of endomorphin-2 in the dorsal horn of the medulla and spinal cord of the rat. Neuroreport 10, 3857 /3860. Brown, D.L., Guyenet, P.G., 1985. Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ. Res. 56, 359 /369. Chen, Z., Hedner, J., Hedner, T., 1996. Substance P induced respiratory excitation is blunted by delta opioid receptor
T. Lonergan et al. / Respiratory Physiology & Neurobiology 138 (2003) 165 /178 specific opioids in the rat medulla oblongata. Acta Physiol. Scand. 157, 165 /173. Connor, M., Schuller, A.G.P., Pintar, J.E., Christie, M.J., 1999. Mu opioid receptor modulation of calcium channel current in periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR1. Br. J. Pharmacol. 126, 1553 / 1558. Czapla, M.A., Gozal, D., Alea, O.A., Beckerman, R.C., Zadina, J., 2000. Differential cardiorespiratory effects of endomorphin 1, endomorphin 2, DAMGO and morphine. Am. J. Respir. Crit. Care Med. 162, 994 /999. Denavit-Saubie´, M., Champagnat, J., Zieglgansberger, W., 1978. Effects of opiates and methionine /enkephalin on pontine and bulbar respiratory neurones of the cat. Brain Res. 155, 55 /67. Ding, Y., Kaneko, T., Nomura, S., Mizuno, N., 1996. Immunohistochemical localisation of mu-opioid receptors in the central nervous system of the rat. J. Comp. Neurol. 367, 375 /402. Ellenberger, H.H., Feldman, J.L., 1990. Brainstem connections of the rostral ventral respiratory group of the rat. Brain Res. 513, 35 /42. Ezure, K., 1990. Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog. Neurobiol. 35, 429 /450. Ezure, K., Manabe, M., 1988. Decrementing expiratory neurons of the Bo¨tzinger complex. II. Direct inhibitory synaptic linkage with ventral respiratory group neurons. Exp. Brain Res. 72, 159 /166. Fung, M.L., Wang, W., St-John, W.M., 1994. Medullary loci critical for expression of gasping in adult rats. J. Physiol. (London) 480, 597 /611. Fung, M.L., Huang, Q., Zhou, D., St-John, W.M., 1997. Morphology and connections of neurons in the gasping centre of adult rats. Neuroscience 76, 1237 /1244. Gaytan, S.P., Calero, F., Nu´n˜ez-Abades, P.A., Morillo, A.M., Pasaro, R., 1997. Pontomedullary efferent projections of the ventral respiratory neuronal subsets of the rat. Brain Res. Bull. 42, 323 /334. Goldstein, A., Naidu, A., 1989. Multiple opioid receptors: ligand selectivity profiles and binding site signatures. Mol. Pharmacol. 36, 265 /272. Gray, P.A., Rekling, J.C., Bocchario, M., Feldman, J.L., 1999. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the pre-Bo¨tzinger complex. Science 286, 1566 /1568. Greer, J.J., Carter, J.E., Al-Zubaidy, Z., 1995. Opioid depression of respiration in neonatal rats. J. Physiol. (London) 485, 845 /855. Hassen, A.H., Feuerstein, G., Faden, A.I., 1984. Selective cardiorespiratory effects mediated by mu opioid receptors in the nucleus ambiguus. Neuropharmacology 23, 407 /415. Horvath, G., 2000. Endomorphin-1 and endomorphin-2: pharmacology of the selective endogenous mu opioid receptor agonists. Pharmacol. Ther. 88, 437 /463. Hurle, M.A., Mediavilla, A., Florez, J., 1985. Differential respiratory patterns induced by opioids applied to the
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ventral medullary and dorsal pontine surfaces of cats. Neuropharmacology 24, 597 /606. Janczewski, W.A., Feldman, J.L., 2000. Microinjection of a mu opioid receptor (mOR) agonist into the pre-Bo¨tzinger complex (pre-Bo¨tC) abolishes automatic breathing movements in adult rats. Soc. Neurosci. Proc. 409, 5. Jiang, C., Lipski, J., 1990. Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Bo¨tzinger complex of the cat. Exp. Brain Res. 81, 639 /648. Johnson, S.M., Smith, J.C., Feldman, J.L., 1996. Modulation of respiratory rhythm in vitro: role of Gi/o protein mediated mechanisms. J. Appl. Physiol. 80, 2120 /2133. Llewellyn-Smith, I.J., Minson, J.B., Pilowsky, P.M., 1992. Retrograde tracing with cholera toxin B-gold or immunocytochemically detected cholera toxin-B in central nervous system. In: Conn, P.M. (Ed.), Methods in Neuroscience, vol. 8. Academic Press, San Diego, pp. 180 /201. Milner, T.A., Drake, C.T., Aicher, S., 2001. Cellular relations between mu opioid receptive, GABAergic and reticulospinal neurons in the rostroventrolateral medulla. Brain Res. 917, 1 /14. Miyawaki, T., Goodchild, A.K., Pilowsky, P.M., 2002. Activation of mu-opiate receptors in rat ventrolateral medulla selectively blocks baroreceptor reflexes while activation of delta-receptors blocks somato-symapthetic reflexes. Neuroscience 109, 133 /144. Morin-Surun, M.P., Boudinot, E., Gacel, G., Champagnat, J., Roques, B.P., Denavit-Saubie´, M., 1984. Different effects of mu and delta opiate agonists on respiration. Eur. J. Pharmacol. 98, 235 /240. Murakoshi, T., Suzue, T., Tamai, S., 1985. A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat. Br. J. Pharmacol. 86, 95 /104. Negri, L., Lattanzi, R., Tabacco, F., Melchiorri, P., 1998. Respiratory and cardiovascular effects of the mu opioid receptor agonist [lys 7]dermorphin in awake rats. Br. J. Pharmacol. 124, 345 /355. Nu´n˜ez-Abades, P.A., Pasaro, R., Bianchi, A.L., 1991. Localisation of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat. Brain Res. 568, 165 /172. Nu´n˜ez-Abades, P.A., Morillo, A.M., Pasaro, R., 1993. Brainstem connections of the rat ventral respiratory subgroups: afferent projections. J Autonom. Nerv. Syst. 42, 99 /118. O’Neal, M.H., Halat, T.J., Edelman, N., Solomon, I.C., 2000. Co-expression of gap-junction proteins with mu-opioid and substance-P receptors in the pre-Bo¨tzinger complex of the neonatal and adult rat. Soc. Neurosci. Proc. 347, 5. Paton, J.F.R., Richter, D.W., 1995. Role of fast inhibitory synaptic mechanisms in respiratory rhythm generation in the maturing mouse. J. Physiol. (London) 484, 505 /521. Paton, J.F.R., Ramirez, J.M., Richter, D.W., 1994. Mechanisms of respiratory rhythm generation change profoundly during early life in mice and rats. Neurosci. Lett. 170, 167 / 170.
178
T. Lonergan et al. / Respiratory Physiology & Neurobiology 138 (2003) 165 /178
Pazos, A., Florez, J., 1983. Interaction of naloxone with mu and delta opioid agonists on the respiration of rats. Eur. J. Pharmacol. 87, 309 /314. Pazos, A., Florez, J., 1984. A comparative study in rats of the respiratory depression and analgesia induced by mu and delta opioid agonists. Eur. J. Pharmacol. 99, 15 /21. Pilowsky, P.M., Jiang, C., Lipski, J., 1990. An intracellular study of respiratory neurons in the rostral ventrolateral medulla of the rat and their relationship to catecholamine containing neurons. J. Comp. Neurol. 301, 604 /617. Ptak, K., Hilaire, G., 1999. Central respiratory effects of substance P in neonatal mice: an in vitro study. Neurosci. Lett. 266, 189 /192. Rondouin, G., Boudinot, E., Champagnat, J., Denavit-Saubie´, M., 1981. Effects of sulphonated leu-enkephalin applied iontophoretically to cat respiratory neurons. Neuropharmacology 20, 963 /967. St.-Jaques, R., St.-John, W.M., 1999. Transient, reversible apnoea following ablation of the pre-Bo¨tzinger complex in rats. J. Physiol. (London) 520, 303 /314. Sun, Q.-J., Goodchild, A.K., Chalmers, J.P., Pilowsky, P.M., 1998. The pre-Bo¨tzinger complex and phase-spanning neurons in the adult rat. Brain Res. 809, 204 /213. Swanson, L.W. (Ed.), Brain Maps: Structure of the Rat Brain, second ed.. Elselvier, Amsterdam 1998. Takita, K., Herlenius, E.A.P., Lindhal, S.G.E., Yamamoto, Y., 1997. Actions of opioids on respiratory activity via the activation of brainstem mu, delta and kappa receptors; an in vitro study. Brain Res. 778, 233 /241.
Tian, G.-F., Peever, J.H., Duffin, J., 1998. Bo¨tzinger complex expiratory neurons monosynaptically inhibit phreninc motoneurons in the decerebrate rat. Exp. Brain Res. 122, 149 / 156. Tian, G.-F., Peever, J.H., Duffin, J., 1999a. Bo¨tzinger complex, bulbospinal expiratory neurons monosynaptically inhibit ventral group respiratory neurons in the decerebrate rat. Exp. Brain Res. 124, 173 /180. Tian, G.-F., Peever, J.H., Duffin, J., 1999b. Mutual inhibition between Bo¨tzinger complex bulbospinal expiratory neurons detected with cross correlation in the decerebrate rat. Exp. Brain Res. 125, 440 /446. Wang, H., Stornetta, R.L., Rosin, D.L., Guyenet, P.G., 2001. Neurokinin-1 receptor immunoreactive neurons of the ventral respiratory group in the rat. J. Comp. Neurol. 434, 128 /146. Zadina, J., Hackler, L., Ge, L.-J., Kastin, A.J., 1997. A potent and selective endogenous agonist for the mu opiate receptor. Nature 386, 499 /502. Zadina, J., Martin-Schild, S., Gerall, A.A., Kastin, A.J., Hackler, L., Ge, L.-J., Zhang, X., 1999. Endomorphins: novel endogenous mu-opiate receptor agonists in regions of high mu-opiate receptor density. Ann. N.Y. Acad. Sci. 897, 136 /144. Zhang, W., Elsen, F., Barnbrock, A., Richter, D.W., 1999. Postnatal development of GABAb receptor-mediated modulation of voltage-activated calcium currents in mouse brainstem neurons. Eur. J. Neurosci. 11, 2332 /2342.
Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity Tina Lonergan a,b,1, Ann K. Goodchild b, Macdonald J. Christie a, Paul M. Pilowsky a,b,* b
a Department of Pharmacology, University of Sydney, Camperdown, NSW 2006, Australia Hypertension and Stroke Research Laboratories, Department of Physiology, University of Sydney and Department of Neurosurgery, Royal North Shore Hospital, Ground Floor, Block 3, St. Leonards, NSW 2065, Australia
Accepted 4 June 2003
Abstract Anatomical and in vitro studies suggest that mu opioid receptors (MOR) on pre-Bo¨tzinger complex neurons are responsible for opioid induced respiratory depression (Grey et al., Science 286 (1999) 1566). However, mu opioid agonists injected in vivo, in other regions of the ventral respiratory group (VRG), produce respiratory depression, suggesting that opioids are widely distributed in the VRG. We therefore re-examined the distribution of the MOR in the ventral medulla and found MOR-immunoreactive neurons and terminals in all subdivisions of the VRG. Furthermore, we determined, in rats, the effects of a MOR agonist (endomorphin-1, 10 mM, 60 nl, unilateral), microinjected into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 produced changes in phrenic nerve frequency and amplitude, throughout the VRG. Unexpectedly, endomorphin-1 microinjected into the Bo¨tzinger and pre-Bo¨tzinger complexes consistently increased phrenic nerve frequency. These results support the widespread distribution of MOR in the VRG and also indicate that endomorphin-1, a postulated endogenous ligand, may differentially regulate respiration. # 2003 Elsevier B.V. All rights reserved. Keywords: Brainstem; Pre-Bo¨tzinger region; Mu opioid receptors; Control of breathing; Respiratory depression; Mammals; Rat; Receptors; Mu opioid
1. Introduction The mu opioid receptor (MOR) is implicated in the respiratory depressant actions of opioids in the medulla. Early, in vivo, work demonstrated that
intracerebroventricular (Pazos and Florez, 1983; Hurle et al., 1985), or ventral medullary surface application (Hurle et al., 1985) of MOR selective agonists caused respiratory depression, however, the exact neuronal structures involved in MOR
* Corresponding author. Tel.: /61-2-9926-8080; fax: /61-2-9926-6483. http://www.physiol.usyd.edu.au/ /pilowsky/. E-mail address: [email protected] (P.M. Pilowsky). 1 Formerly Stasinopoulos. 1569-9048/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1569-9048(03)00173-3
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induced respiratory depression remain loosely defined. Gray et al. (1999) suggested that opioid respiratory depression is due to inhibition of rhythm generating neurons in the pre-Bo¨tzinger complex and that MOR-immunoreactivity (ir) is exclusively found in that area. While the involvement of the pre-Bo¨tzinger complex in MOR induced respiratory depression is well established in neonatal in vitro preparations (Murakoshi et al., 1985; Greer et al., 1995; Johnson et al., 1996; Ballanyi et al., 1997; Takita et al., 1997; Gray et al., 1999), other structures may be involved in the adult, in vivo. In adult rats and cats, iontophoresis of opioids onto respiratory units throughout the ventral respiratory group (VRG) inhibited their activity (Denavit-Saubie´ et al., 1978; Rondouin et al., 1981) and microinjection of the selective MOR agonist, DAMGO, into the rostral VRG (rVRG) or the nucleus ambiguus (NA), depressed respiration (Hassen et al., 1984; Chen et al., 1996). These data suggest that the MOR is widely distributed in the VRG of the adult rat, and that it modulates the activity of a wide range of respiratory neurons. Therefore the aims of this study were, firstly, to re-examine the distribution of the MOR in the ventral medulla. Secondly, to determine the effects of microinjection of endomorphin1, into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 was chosen as it is suggested to be an endogenous ligand for the MOR (Zadina et al., 1997). Furthermore, unlike other endogenous peptides, which are only 10 / more selective for the MOR over the delta opioid receptor (DOR) (Goldstein and Naidu, 1989), endomorphin-1 is more than 4000 / selective for the MOR over the DOR (Zadina et al., 1997).
approved by the Animal Care and Ethics Committee of the Royal North Shore Hospital. 2.1. Immunohistochemistry Rats (n/3) were anaesthetised (sodium pentobarbital, 100 mg/kg, i.p.) and intracardially perfused with DMEM (Sigma), followed by 4% formaldehyde in 0.1 M phosphate buffer (PB), at pH 7.4. Brainstems were removed and post-fixed for 1.5 h and serial 50 mm sections were cut using a vibrating microtome. The sections were washed for 30 min in 50% ethanol, followed by 3/30 min washes in tris-phosphate buffered saline (TPBS; Tris /HCl 10 mM, sodium phosphate buffer 10 mM, 0.9% NaCl, pH 7.4). Sections were incubated in a rabbit anti-MOR1 antibody (1:20 000, Incstar) and 5% normal horse serum (NHS) in TPBS, for 72 h. This was followed by overnight incubations in biotinylated-donkey anti rabbit F(ab?)2 fragments (1: 500, Jackson: 711-066-152) and 2% NHS in TPBS, then ExtrAvidin-HRP in TPBS (1:1000, Sigma). MOR-ir was revealed using a nickel intensified, glucose-oxidase peroxidase reaction (Llewellyn-Smith et al., 1992). Inclusion of the peptide (10 mg/ml) against which the antibody was raised, abolished MOR-ir, suggesting the antibody was specific. Washes were performed between incubations (TPBS, 3 /30 min), at room temperature. Sections were serially mounted, dehydrated, and coverslipped using Ultramount (Fronine Pty Ltd, Australia). Sections were viewed using a Leica DML microscope and images were obtained using a Spot2 digital camera (Diagnostic Instruments), contrast and brightness adjusted for using Spot2 proprietary software. 2.2. Microinjections
2. Experimental procedures Adult male Sprague/Dawley rats (200 /500 g) were used. Experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes as endorsed by the National Health & Medical Research Council of Australia. Protocols were
Rats (n/8) were anaesthetised with urethane (1.3 g/kg, i.p.), additional doses given as required (0.2 g/kg, i.v.). Atropine sulphate (0.15 mg/kg, i.p.) was administered to reduce airway secretions. The trachea, left femoral artery and vein were cannulated. The cervical vagus, phrenic nerve and the facial nerve were exposed. Rats were placed in a stereotaxic frame, ventilated, paralysed, bilaterally
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vagotomised and the dorsal surface of the medulla exposed. End tidal CO2 was monitored and maintained between 4 and 5%. The vagus and phrenic nerves were placed on bipolar silver electrodes for stimulation and recording, respectively. Bipolar stimulating electrodes were hooked onto the facial nerve. Phrenic nerve discharge was acquired at a gain of 20 000 /50 000 (AC-coupled preamplifier and amplifier, CWE), band pass filtered (10 /1 kHz), digitised and sampled at 2000 Hz (1401-plus, CED, UK). Multibarrel glass pipettes were constructed and filled with 3 M NaCl, endomorphin-1 (10 mM, in 0.01 M PBS, pH 7.4, Chiron Technologies, Australia) and albumin/colloidal gold (20% in 0.01 M PBS, pH 7.4, Sigma). The location of the facial nucleus (VIIn), compact formation of the nucleus ambiguus (NAc) and respiratory field activity were mapped by antidromic stimulation of the facial and vagus nerve, as previously described (Brown and Guyenet, 1985; Pilowsky et al., 1990; Sun et al., 1998). The extracellular signal was acquired with a 10 / gain (Axoclamp 2B, Axon Instruments), further amplified (200 /1000/) and band pass filtered (30 /3 kHz, AC Bioamplifier, CWE), digitised and sampled at 2000 Hz (CED 1401plus). Unilateral microinjections of endomorphin-1 (60 nl) were made in regions where respiratory field activity could be recorded. The effect on phrenic nerve discharge and mean arterial pressure (MAP) was monitored for at least 30 min, following each injection. Injections of colloidal gold (60 nl) were made following each endmorphin-1 injection. At the end of the experiment, the rat was euthanised (3 M KCl, i.v.) and the brainstem removed and fixed overnight. Sections (100 mm) were cut using a vibrating microtome and processed for gold injection sites using a silver enhancer kit (Sigma). Sections were mounted on gelatinised slides, dehydrated, counterstained using cresyl violet, and coverslipped.
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2.3. Data analysis 2.3.1. Analysis of phrenic nerve discharge Phrenic nerve activity was analysed offline using SPIKE2 software (CED). Raw phrenic nerve activity was rectified and integrated as a moving average with a 20 msec time constant. Waveform averages and mean frequencies of phrenic nerve discharge were obtained over 2 min periods. The inspiratory time, expiratory time, respiratory cycle time and peak amplitude of phrenic nerve discharge were calculated from waveform averages. Pre-drug values were compared to post-drug values at the following times: 1, 5, 10, 15, 20, 25 and 30 min post-injection. For most of the injection sites, phrenic nerve activity was no longer significantly different from control at 30 min (significance was taken at /15% difference from control). When the effects were still significant at 30 min, measurements were taken approximately every 10 min until the changes were no longer significantly different to control. Apnoea was defined as the absence of phrenic nerve discharge for periods greater than 3 sec. 2.3.2. Determination of location of injection sites The centre of injection sites was determined and plotted onto brainstem maps (Swanson, 1998) with reference to the caudal border of the VIIn (bregma /12.75 mm).
3. Results 3.1. Immunohistochemistry Intense MOR-ir was observed in many regions of the medulla, including the area postrema, dorsal motonucleus of the vagus, solitary tract and nucleus, spinal trigeminal nucleus, inferior olive, and the NAc, as previously reported (Arvidsson et al., 1995; Ding et al., 1996). Less intense MOR-ir was present throughout the medulla, including the VRG (Fig. 1). Small numbers of weakly MOR-ir neurons were found throughout the rostrocaudal extent of the VRG (Fig. 1), while MOR-ir fibres and terminals were more abundant (Fig. 1). There appeared to be no difference in the number of
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Fig. 1. Distribution of MOR-ir in the region of the VRG. (A) cVRG, (B) rVRG, (C) pre-Bo¨tzinger complex, (D) Bo¨tzinger complex. The boxed areas in the low power images are shown in the high power images on the right. In the region of the VRG, numerous MORir neurons(*), fibres (arrow) and varicosities (arrow heads) were observed. Intense MOR-ir was observed in the area postrema, dorsal motonucleus of the vagus, solitary tract and nucleus, spinal trigeminal nucleus, inferior olive, the NAc and vagal afferents in the rostral medulla. Scale bars are 100 mm.
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MOR-ir neurons in the region of the pre-Bo¨tzinger complex compared to the adjacent Bo¨tzinger complex and rVRG. A group of intensely labelled MOR-ir neurons and fibres were observed dorsomedial to where the pre-Bo¨tzinger is thought to lie (Fig. 1C). The rostrocaudal extent of these neurons overlaps with that of the pre-Bo¨tzinger, although these neurons appear to extend rostrally for another 200 mm. 3.2. Microinjection of endomorphin-1 into the VRG Endomorphin-1 was microinjected into 23 sites in the ventral medulla, spanning the Bo¨tzinger complex, pre-Bo¨tzinger complex and rVRG (defined by rostrocaudal location and respiratory field activity at the site of injection (Sun et al., 1998). Figs. 2/4 show the injection sites and effects of endomorphin-1 on phrenic nerve discharge frequency (Fig. 2), amplitude (Fig. 3) and MAP (Fig. 4). Injection of albumin-colloidal gold suspended in the same vehicle as endomorphin-1, did not alter phrenic nerve activity or MAP, confirming that the effects seen were due to the actions of endomorphin-1. 3.2.1. Bo ¨ tzinger complex A total of six injections of endomorphin-1 were made into the Bo¨tzinger complex, in sites of expiratory (5) or E-I field activity (1), 100/700 mm caudal to the caudal border of the VIIn (Fig. 2i, Fig. 5). Endomorphin-1 injected into the Bo¨tzinger complex increased phrenic nerve frequency by 16 /30%, within the first 10 min, at 4/6 sites, (Fig. 2i, Fig. 5A). The responses usually disappeared within 20 min (Fig. 2i). At one site, endomorphin-1 elicited a decrease in phrenic nerve frequency that was followed by apnoea (Fig. 5B). The changes in frequency occurred as a result of shortening (Fig. 5A) or lengthening (Fig. 5B) of expiratory period. Endomorphin-1 attenuated phrenic nerve amplitude by 16/100%, at 5/8 sites (Fig. 3i). At 2 of these sites, apnoea occurred (e.g. Fig. 5B). At one site, endomorphin-1 produced an increase in amplitude, with a maximal increase of 37% at 20 min post-injection. MAP was altered by
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endomorphin-1 at 2/6 sites in the Bo¨tzinger complex (Fig. 4i), injection at one site producing a depressor response, the other site producing a pressor response. 3.2.2. Pre-Bo ¨ tzinger complex A total of 10 microinjections of endomorphin-1 were made into the pre-Bo¨tzinger complex, in sites of inspiratory (6), expiratory (3) or E-I field activity (1), 0.75 /1.25 mm caudal from the caudal border of the VIIn (Fig. 2ii, iii, Fig. 3ii, iii, Fig. 4ii, iii). A reduction in frequency was only observed at 1/10 injection sites in the pre-Bo¨tzinger complex. At 8/10 sites, endomorphin-1 increased the frequency of phrenic nerve discharge by 16 /45% (Fig. 2ii, iii). The increase in frequency usually lasted for about 10 min (5/8 sites), but in some cases (3/8) lasted for 20/30 min (e.g. Fig. 6A). Changes in frequency occurred as a result of changes in both the inspiratory and expiratory period (e.g. Fig. 6A). Injection of endomorphin-1 at 7/10 sites reduced phrenic nerve amplitude by 16/50% (e.g. Fig. 6A) and the response lasted anywhere between 10 /30 min, (Fig. 3ii, iii). Injection of endomorphin-1 at 3/10 sites increased phrenic nerve amplitude by 16/30%. Excitatory and inhibitory effects on amplitude could not be distinguished by location or type of respiratory field activity at the site of injection. Endomorphin1 evoked depressor responses at 4/10 sites (Fig. 4ii, iii). 3.2.3. rVRG A total of seven injections of endomorphin-1 were made into the rVRG, in sites of inspiratory field activity, 1.30 /2.05 mm caudal from the caudal border of the VIIn (Fig. 2iv, v). Endomorphin-1 only produced effects on frequency at 3/7 sites in the rVRG. Injection of endomorphin-1 at one site increased frequency by 32% in the first 10 min. Endomorphin-1 injected at 2 sites produced a decrease in frequency, lasting 10/20 min, apnoea occurring at one of these sites (Fig. 6B). The changes in frequency caused by endomorphin-1 were largely due to changes in expiratory period. Endomorphin-1 produced a decrease in phrenic nerve amplitude at 6/7 sites (e.g. Fig. 6B). This fall in amplitude lasted between
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Fig. 2. Time course of effects (left to right) of unilateral microinjections of endomorphin-1 (10 mM, 60 nl), into different rostrocaudal levels of the VRG on phrenic nerve discharge frequency. Each circle or square represents an injection site and the increasing darkness and size relates to increasing changes that occurred during that particular time period.
10 /60 min (Fig. 3iv, v) and elicited a depressor response at 2/7 sites (Fig. 4iv).
4. Discussion This study demonstrates MOR-ir on neuronal perikarya and terminals throughout the rostrocaudal extent of the VRG. Supporting the widespread distribution of MOR-ir in the VRG, endomorphin-1 produced changes in both phrenic nerve amplitude and frequency in all regions of the VRG tested.
4.1. Relationship between MOR-ir and subdivisions of the VRG A small number of weakly MOR-ir neurons were observed throughout the ventral respiratory column. The number of MOR-ir neurons observed was far less than the number of NK-1-ir neurons that have been reported in the same region (Wang et al., 2001). The low level of MOR-ir in the VRG suggests that either the respiratory effects of opioids are mediated by a small number of receptors or that the MOR antibody is not sensitive enough to detect all the receptors in the area.
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Fig. 3. Same data as in Fig. 2, but plotting changes in phrenic nerve amplitude.
Gray et al. (1999) proposed that MOR-ir was confined to the pre-Bo¨tzinger complex and therefore could be used as neurochemical/anatomical marker of the pre-Bo¨tzinger complex (Gray et al., 1999). However, in this study MOR-ir was also found in the adjacent Bo¨tzinger complex, rVRG and cVRG. The reason for the discrepancy between these studies is not clear. While the same antibody was used in both studies, female rats and mice were used in Gray’s study, and the exact age was not specified. This may bear on the results since it was shown that MOR-ir decreases in the pre-Bo¨tzinger with age (O’Neal et al., 2000). Furthermore, thin cryostat sections and tyramine signal amplification was used to amplify MOR-ir in some of the experiments of Gray et al. (1999).
The differences in processing may explain the enhanced detection of MOR-ir in the pre-Bo¨tzinger; however, they do not explain why comparable MOR-ir was not detected in the adjacent Bo¨tzinger and rVRG by Gray et al. (1999). The pharmacological findings that MOR activation in the Bo¨tzinger and rVRG produced changes in phrenic nerve activity supports the immunohistochemical evidence presented in this study. The widespread distribution of MOR-ir neurons and terminals in the ventral medulla suggests that opioids, both endogenous and exogenous may directly inhibit, or inhibit afferent inputs to, respiratory neurons throughout the VRG. However, the usual physiological function of the MORir structures detected is unknown and they may
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Fig. 4. Same data as in Fig. 2, but plotting changes in MAP.
subserve respiratory, cardiovascular, or other functions. For example, C1 and sympathoexcitatory neurons in the rostral ventrolateral medulla are MOR-ir or receive synaptic inputs that are MOR-ir (Aicher et al., 2001a,b; Milner et al., 2001). The present study defined for the first time, a group of intensely stained MOR-ir neurons that were located dorsomedially to the MOR-ir neurons defined by Gray et al. (1999). The function of these MOR-ir neurons is unknown. Interestingly, the region where these MOR-ir neurons are located corresponds well with the gasping centre described by St.-John and colleagues in the rat (Fung et al., 1994, 1997; St.-Jaques and St.-John, 1999). The region also overlaps with sites that produced apnoea when DAMGO (a MOR ago-
nist) was microinjected into the rat (Hassen et al., 1984). Together, these data suggest that the medial MOR-ir neurons may be a part of the respiratory network. 4.2. Effects of endomorphin-1 in the VRG 4.2.1. Technical considerations Caution must be taken in extrapolating these results to the intact, conscious animal since the use of anaesthesia and vagotomy may remove tonically active inputs that may be modulated by the activation of MOR. Furthermore, unilateral injections may produce different responses compared to bilateral microinjections, since the contralateral side of the VRG receives inputs from the ipsilateral side (Ezure, 1990; Jiang and Lipski, 1990;
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Fig. 5. Effects of endomorphin-1 (10 mM, 60 nl, unilateral) microinjected into two sites in the Bo¨tzinger complex. (A) Endomorphin-1 injected into this site of expiratory field activity, produced a small reduction in phrenic nerve amplitude and a 30% increase in phrenic nerve frequency during the first 10 min. The increase in frequency was due to a decrease in expiratory period (see waveform average). (B) Endomorphin-1 injected into this site reduced phrenic nerve amplitude and frequency, leading to apnoea. MAP was initially reduced and then became erratic and did not return to normal until phrenic nerve activity recovered. The apnoea was due to a prolongation in expiratory time (see waveform average).
Nu´n˜ez-Abades et al., 1991; Gaytan et al., 1997; Wang et al., 2001) and may act to restore phrenic nerve activity. No previous study has microinjected endomorphin-1 into the brainstem, so a dose of 10 mM was used. Activation of the DOR by endomorphin-1 at the dose used, cannot be ruled out in these experiments. It has also been suggested that endomorphin-1 is a partial agonist in some tissues (Connor et al., 1999; Horvath, 2000), however the partial-agonist activity of endomorphin-1 in the medulla is undefined. Antagonists were not used in these experiments since it was observed that
desensitisation occurred following repeated administration of endomorphin-1 in the same site.
4.2.2. Comparison with other studies Studies where opioids were applied to the ventral/dorsal brainstem surface, microinjected intracerebroventricularly or into the medulla, report similar time courses to this study, with the time to peak ranging from 1 to 30 min and duration of effect ranging from 30 min to more than 2 h (Pazos and Florez, 1983, 1984; MorinSurun et al., 1984; Hurle et al., 1985; Chen et al.,
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Fig. 6. Effects of endomorphin-1 (10 mM, 60 nl, unilateral) microinjected into a site of expiratory field activity in the pre-Bo¨tzinger complex (A) and a site of inspiratory field activity in the rVRG (B). Endomorphin-1 produced a large reduction of phrenic nerve amplitude at both sites but produced an increase (25%) of phrenic nerve activity in the pre-Bo¨tzinger complex, but a decrease in frequency, that led to apnoea, in the rVRG. At both sites, the changes in frequency were due to alteration of the expiratory period (see waveform averages). There was little change in MAP at these sites.
1996; Takita et al., 1997; Janczewski and Feldman, 2000). In vivo microinjection studies using selective MOR agonists in the VRG are rare. No single study has mapped the effects of MOR agonists in different subdivisions of VRG. Instead, they focused on particular areas. For example, DAMGO microinjected unilaterally into the region of the NAc in the rat produced a decrease in respiratory frequency, instead of the increase seen
with endomorphin-1 in this study (Hassen et al., 1984). An increase in respiratory frequency was observed in several studies following intravenous administration of specific MOR agonists in conscious animals (Negri et al., 1998; Czapla et al., 2000), however activation of MOR in other regions of the brain may have contributed. DAMGO in the rVRG of the rat produced a decrease in frequency and amplitude (Chen et al., 1996), similar to endomorphin-1 in this study.
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Surprisingly, in the present study, apnoea was never evoked following microinjection of endomorphin-1 into the pre-Bo¨tzinger complex. This is contrary to in vitro (Greer et al., 1995; Johnson et al., 1996; Ballanyi et al., 1997; Takita et al., 1997; Gray et al., 1999) and in vivo studies (Janczewski and Feldman, 2000) that attribute opioid respiratory depression to MOR-induced inhibition of rhythm generating neurons in the pre-Bo¨tzinger complex. The differences between the current data and in vitro studies may reflect maturational changes (Paton et al., 1994; Paton and Richter, 1995; Ptak and Hilaire, 1999; Zhang et al., 1999), the absence of afferent inputs in vitro and the presence of anaesthetic in vivo. The differences seen between other in vivo experiments and the present study could reflect the difference in the experimental model used, for example, bilateral vs. unilateral microinjections, different anaesthetics (pentobarbitone vs. urethane), vagotomised vs. vagus intact, artificially ventilated vs. spontaneously breathing, and finally, the use of different ligands. 4.2.3. Mechanism of action of endomorphin-1 in the VRG 4.2.3.1. Bo ¨ tzinger complex. The increase in phrenic nerve discharge frequency following endomorphin-1 injections into the Bo¨tzinger complex may have resulted from an inhibition of excitatory inputs to Bo¨tzinger neurons or hyperpolarisation of Bo¨tzinger neurons, such that expiration was terminated earlier. The depression of phrenic nerve amplitude following endomophin-1 was possibly due to removal of inhibitory inputs to Bo¨tzinger neurons resulting in increased inhibitory activity to pattern generating neurons, inspiratory premotoneurons or phrenic motoneurons (Ezure and Manabe, 1988; Ezure, 1990; Jiang and Lipski, 1990; Tian et al., 1998, 1999a,b). 4.2.3.2. Pre-Bo ¨ tzinger complex. The increase in phrenic nerve discharge frequency following microinjection of endomorphin-1 into the pre-Bo¨tzinger complex may be due to the removal of inhibitory inputs to the pre-Bo¨tzinger, e.g. from
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the Bo¨tzinger complex. Since the increase in frequency was due to changes in both expiratory and inspiratory timing, activation of the MOR by endomorphin-1 in the pre-Bo¨tzinger affects mechanisms that determine both the inspiratory on and off switches. This is in contrast to the Bo¨tzinger complex and rVRG, where the MOR largely affected the length of expiration and thus the inspiratory on-switch. The observation of mixed effects on phrenic nerve amplitude following endomorphin-1 microinjection into the pre-Bo¨tzinger complex suggests that the MOR is found on more than one type of neuron in the pre-Bo¨tzinger complex, or on more than one afferent input to the pre-Bo¨tzinger. This is in contrast to the theory proposed by Gray et al. (1999) that the MOR is only found on type-I pre-I neurons. Indeed the immunohistochemical studies described here show that MOR-ir is observed on both terminals and neurons throughout the VRG.
4.2.3.3. rVRG. In the rVRG endomorphin-1 usually reduced phrenic nerve frequency suggesting a differentiation in the function of the MOR (or endomorphin-1) in the modulation of rhythm generation in the rVRG compared to the Bo¨tzinger and pre-Bo¨tzinger complexes. The depression of both phrenic nerve amplitude and frequency in the rVRG indicates that the role of the MOR in the VRG is to suppress the excitatory function of the bulbospinal neurons (either by inhibiting excitatory inputs and/or by hypopolarising bulbospinal neurons). Indeed Lalley (personal communication) has shown that DAMGO hyperpolarises augmenting bulbospinal inspiratory neurons in vivo. The occurrence of apnoea at one site further supports this idea. In all the regions discussed above there is also a possibility that some of the effects produced on phrenic nerve activity were due to activation of opioid receptors on the distal dendrites of respiratory neurons located in other regions of the VRG. For example, effects observed in the Bo¨tzinger complex may be due to actions on the dendrites of pre-Bo¨tzinger neurons located in this area.
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4.2.4. Effect of endomorphin-1 on MAP MAP was not affected greatly by injections of endomorphin-1, despite the recent identification of pre- and post-synaptic MOR-ir on functionally identified presympathetic neurons (Aicher et al., 2001a,b; Milner et al., 2001). The lack of effect may be due to the partial agonist activity of endomorphin-1. Other studies in our lab have shown that DAMGO, a full MOR agonist caused a significant depressor response when injected in the rostral ventrolateral medulla (Miyawaki et al., 2002).
lead compound in the search for opiates with less respiratory depressant effects than current agents. The multitude of responses and the location of the MOR in more than one region of the VRG suggest that the MOR plays a diverse role in the VRG. The effect of activation of the MOR, on central respiratory output is likely to depend on the endogenous ligand released, the location of release and the afferent inputs and respiratory neurons affected.
Acknowledgements 4.3. Endogenous endomorphin The mRNA and precursor protein that produces endomorphin-1 is unidentified. Endomorphin-1-like-ir terminals are distributed throughout the rat brain, including the brainstem (Zadina et al., 1999). Endomorphin-1-like-ir cell bodies are found in the hypothalamus and nucleus tractus solitarius (NTS), (Zadina et al., 1999), with a distribution akin to that of b-endorphin. The NTS could be a source of endomorphin-1 inputs to the VRG, as it projects to the VRG (Ellenberger and Feldman, 1990; Nu´n˜ez-Abades et al., 1993). Endomorphin-1-like-ir does not appear at birth and only matures at 21 days (Barr and Zadina, 1999), indicating that the function and activity of the MOR may change as the respiratory network matures. 4.4. Conclusion We have shown that MOR-ir is present throughout the VRG. Furthermore, activation of the MOR, by microinjection of endomorphin-1, into the Bo¨tzinger, pre-Bo¨tzinger or rVRG results in profound changes in both phrenic nerve frequency (rhythm generation) and phrenic nerve amplitude (pattern formation). However, each subdivision of the VRG produced a unique response to endomorphin-1. Perhaps the most surprising finding was that endomorphin-1 increased phrenic nerve frequency and thus promoted rhythm generation in the Bo¨tzinger and pre-Bo¨tzinger complex. This suggests that endomorphin-1 may be an interesting
Our laboratory is supported by grants from the National Health and Medical Research Council (980077, 211023), National Heart Foundation (GOOS0716) and North Shore Heart Research Foundation. M.J. Christie is supported by the University of Sydney Medical Foundation. T. Lonergan is supported by an Australian Postgraduate Award.
References Aicher, S., Kraus, J.A., Sharma, S., Patel, A., Milner, T.A., 2001a. Selective distribution of mu-opioid receptors in C1 adrenergic neurons and their afferents. J. Comp. Neurol. 433, 23 /33. Aicher, S., Schreihoffer, A.M., Kraus, J.A., Sharma, S., Milner, T.A., Guyenet, P.G., 2001b. Mu-opioid receptors are present in functionally identified sympathoexcitatory neurons in the rat rostral ventrolateral medulla. J. Comp. Neurol. 433, 34 /47. Arvidsson, U., Reidl, M., Chakrabarti, S., Lee, J.-H., Nakano, A.-H., Dado, R.J., Loh, H.H., Law, P.-Y., Wessendorf, M.W., Elde, R., 1995. Distribution and targeting of a mu opioid receptor (MOR1) in brain and spinal cord. J. Neurosci. 15, 3328 /3341. Ballanyi, K., Lalley, P.M., Hoch, B., Richter, D.W., 1997. cAMP-dependent reversal of opioid and prostaglandin mediated depression of the isolated respiratory network in newborn rats. J. Physiol. (London) 504, 127 /134. Barr, G.A., Zadina, J.E., 1999. Maturation of endomorphin-2 in the dorsal horn of the medulla and spinal cord of the rat. Neuroreport 10, 3857 /3860. Brown, D.L., Guyenet, P.G., 1985. Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ. Res. 56, 359 /369. Chen, Z., Hedner, J., Hedner, T., 1996. Substance P induced respiratory excitation is blunted by delta opioid receptor
T. Lonergan et al. / Respiratory Physiology & Neurobiology 138 (2003) 165 /178 specific opioids in the rat medulla oblongata. Acta Physiol. Scand. 157, 165 /173. Connor, M., Schuller, A.G.P., Pintar, J.E., Christie, M.J., 1999. Mu opioid receptor modulation of calcium channel current in periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR1. Br. J. Pharmacol. 126, 1553 / 1558. Czapla, M.A., Gozal, D., Alea, O.A., Beckerman, R.C., Zadina, J., 2000. Differential cardiorespiratory effects of endomorphin 1, endomorphin 2, DAMGO and morphine. Am. J. Respir. Crit. Care Med. 162, 994 /999. Denavit-Saubie´, M., Champagnat, J., Zieglgansberger, W., 1978. Effects of opiates and methionine /enkephalin on pontine and bulbar respiratory neurones of the cat. Brain Res. 155, 55 /67. Ding, Y., Kaneko, T., Nomura, S., Mizuno, N., 1996. Immunohistochemical localisation of mu-opioid receptors in the central nervous system of the rat. J. Comp. Neurol. 367, 375 /402. Ellenberger, H.H., Feldman, J.L., 1990. Brainstem connections of the rostral ventral respiratory group of the rat. Brain Res. 513, 35 /42. Ezure, K., 1990. Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog. Neurobiol. 35, 429 /450. Ezure, K., Manabe, M., 1988. Decrementing expiratory neurons of the Bo¨tzinger complex. II. Direct inhibitory synaptic linkage with ventral respiratory group neurons. Exp. Brain Res. 72, 159 /166. Fung, M.L., Wang, W., St-John, W.M., 1994. Medullary loci critical for expression of gasping in adult rats. J. Physiol. (London) 480, 597 /611. Fung, M.L., Huang, Q., Zhou, D., St-John, W.M., 1997. Morphology and connections of neurons in the gasping centre of adult rats. Neuroscience 76, 1237 /1244. Gaytan, S.P., Calero, F., Nu´n˜ez-Abades, P.A., Morillo, A.M., Pasaro, R., 1997. Pontomedullary efferent projections of the ventral respiratory neuronal subsets of the rat. Brain Res. Bull. 42, 323 /334. Goldstein, A., Naidu, A., 1989. Multiple opioid receptors: ligand selectivity profiles and binding site signatures. Mol. Pharmacol. 36, 265 /272. Gray, P.A., Rekling, J.C., Bocchario, M., Feldman, J.L., 1999. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the pre-Bo¨tzinger complex. Science 286, 1566 /1568. Greer, J.J., Carter, J.E., Al-Zubaidy, Z., 1995. Opioid depression of respiration in neonatal rats. J. Physiol. (London) 485, 845 /855. Hassen, A.H., Feuerstein, G., Faden, A.I., 1984. Selective cardiorespiratory effects mediated by mu opioid receptors in the nucleus ambiguus. Neuropharmacology 23, 407 /415. Horvath, G., 2000. Endomorphin-1 and endomorphin-2: pharmacology of the selective endogenous mu opioid receptor agonists. Pharmacol. Ther. 88, 437 /463. Hurle, M.A., Mediavilla, A., Florez, J., 1985. Differential respiratory patterns induced by opioids applied to the
177
ventral medullary and dorsal pontine surfaces of cats. Neuropharmacology 24, 597 /606. Janczewski, W.A., Feldman, J.L., 2000. Microinjection of a mu opioid receptor (mOR) agonist into the pre-Bo¨tzinger complex (pre-Bo¨tC) abolishes automatic breathing movements in adult rats. Soc. Neurosci. Proc. 409, 5. Jiang, C., Lipski, J., 1990. Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Bo¨tzinger complex of the cat. Exp. Brain Res. 81, 639 /648. Johnson, S.M., Smith, J.C., Feldman, J.L., 1996. Modulation of respiratory rhythm in vitro: role of Gi/o protein mediated mechanisms. J. Appl. Physiol. 80, 2120 /2133. Llewellyn-Smith, I.J., Minson, J.B., Pilowsky, P.M., 1992. Retrograde tracing with cholera toxin B-gold or immunocytochemically detected cholera toxin-B in central nervous system. In: Conn, P.M. (Ed.), Methods in Neuroscience, vol. 8. Academic Press, San Diego, pp. 180 /201. Milner, T.A., Drake, C.T., Aicher, S., 2001. Cellular relations between mu opioid receptive, GABAergic and reticulospinal neurons in the rostroventrolateral medulla. Brain Res. 917, 1 /14. Miyawaki, T., Goodchild, A.K., Pilowsky, P.M., 2002. Activation of mu-opiate receptors in rat ventrolateral medulla selectively blocks baroreceptor reflexes while activation of delta-receptors blocks somato-symapthetic reflexes. Neuroscience 109, 133 /144. Morin-Surun, M.P., Boudinot, E., Gacel, G., Champagnat, J., Roques, B.P., Denavit-Saubie´, M., 1984. Different effects of mu and delta opiate agonists on respiration. Eur. J. Pharmacol. 98, 235 /240. Murakoshi, T., Suzue, T., Tamai, S., 1985. A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat. Br. J. Pharmacol. 86, 95 /104. Negri, L., Lattanzi, R., Tabacco, F., Melchiorri, P., 1998. Respiratory and cardiovascular effects of the mu opioid receptor agonist [lys 7]dermorphin in awake rats. Br. J. Pharmacol. 124, 345 /355. Nu´n˜ez-Abades, P.A., Pasaro, R., Bianchi, A.L., 1991. Localisation of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat. Brain Res. 568, 165 /172. Nu´n˜ez-Abades, P.A., Morillo, A.M., Pasaro, R., 1993. Brainstem connections of the rat ventral respiratory subgroups: afferent projections. J Autonom. Nerv. Syst. 42, 99 /118. O’Neal, M.H., Halat, T.J., Edelman, N., Solomon, I.C., 2000. Co-expression of gap-junction proteins with mu-opioid and substance-P receptors in the pre-Bo¨tzinger complex of the neonatal and adult rat. Soc. Neurosci. Proc. 347, 5. Paton, J.F.R., Richter, D.W., 1995. Role of fast inhibitory synaptic mechanisms in respiratory rhythm generation in the maturing mouse. J. Physiol. (London) 484, 505 /521. Paton, J.F.R., Ramirez, J.M., Richter, D.W., 1994. Mechanisms of respiratory rhythm generation change profoundly during early life in mice and rats. Neurosci. Lett. 170, 167 / 170.
178
T. Lonergan et al. / Respiratory Physiology & Neurobiology 138 (2003) 165 /178
Pazos, A., Florez, J., 1983. Interaction of naloxone with mu and delta opioid agonists on the respiration of rats. Eur. J. Pharmacol. 87, 309 /314. Pazos, A., Florez, J., 1984. A comparative study in rats of the respiratory depression and analgesia induced by mu and delta opioid agonists. Eur. J. Pharmacol. 99, 15 /21. Pilowsky, P.M., Jiang, C., Lipski, J., 1990. An intracellular study of respiratory neurons in the rostral ventrolateral medulla of the rat and their relationship to catecholamine containing neurons. J. Comp. Neurol. 301, 604 /617. Ptak, K., Hilaire, G., 1999. Central respiratory effects of substance P in neonatal mice: an in vitro study. Neurosci. Lett. 266, 189 /192. Rondouin, G., Boudinot, E., Champagnat, J., Denavit-Saubie´, M., 1981. Effects of sulphonated leu-enkephalin applied iontophoretically to cat respiratory neurons. Neuropharmacology 20, 963 /967. St.-Jaques, R., St.-John, W.M., 1999. Transient, reversible apnoea following ablation of the pre-Bo¨tzinger complex in rats. J. Physiol. (London) 520, 303 /314. Sun, Q.-J., Goodchild, A.K., Chalmers, J.P., Pilowsky, P.M., 1998. The pre-Bo¨tzinger complex and phase-spanning neurons in the adult rat. Brain Res. 809, 204 /213. Swanson, L.W. (Ed.), Brain Maps: Structure of the Rat Brain, second ed.. Elselvier, Amsterdam 1998. Takita, K., Herlenius, E.A.P., Lindhal, S.G.E., Yamamoto, Y., 1997. Actions of opioids on respiratory activity via the activation of brainstem mu, delta and kappa receptors; an in vitro study. Brain Res. 778, 233 /241.
Tian, G.-F., Peever, J.H., Duffin, J., 1998. Bo¨tzinger complex expiratory neurons monosynaptically inhibit phreninc motoneurons in the decerebrate rat. Exp. Brain Res. 122, 149 / 156. Tian, G.-F., Peever, J.H., Duffin, J., 1999a. Bo¨tzinger complex, bulbospinal expiratory neurons monosynaptically inhibit ventral group respiratory neurons in the decerebrate rat. Exp. Brain Res. 124, 173 /180. Tian, G.-F., Peever, J.H., Duffin, J., 1999b. Mutual inhibition between Bo¨tzinger complex bulbospinal expiratory neurons detected with cross correlation in the decerebrate rat. Exp. Brain Res. 125, 440 /446. Wang, H., Stornetta, R.L., Rosin, D.L., Guyenet, P.G., 2001. Neurokinin-1 receptor immunoreactive neurons of the ventral respiratory group in the rat. J. Comp. Neurol. 434, 128 /146. Zadina, J., Hackler, L., Ge, L.-J., Kastin, A.J., 1997. A potent and selective endogenous agonist for the mu opiate receptor. Nature 386, 499 /502. Zadina, J., Martin-Schild, S., Gerall, A.A., Kastin, A.J., Hackler, L., Ge, L.-J., Zhang, X., 1999. Endomorphins: novel endogenous mu-opiate receptor agonists in regions of high mu-opiate receptor density. Ann. N.Y. Acad. Sci. 897, 136 /144. Zhang, W., Elsen, F., Barnbrock, A., Richter, D.W., 1999. Postnatal development of GABAb receptor-mediated modulation of voltage-activated calcium currents in mouse brainstem neurons. Eur. J. Neurosci. 11, 2332 /2342.