Respiratory Physiology & Neurobiology 207 (2015) 14–21

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Nociceptin/orphanin FQ slows inspiratory rhythm via its direct effects on the pre-Bötzinger complex Koichi Takita ∗ , Yuji Morimoto Department of Anesthesiology, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-8638, Japan

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Article history: Accepted 4 December 2014 Available online 11 December 2014 Keywords: Nociceptin/orphanin FQ Respiratory rhythm generation Retrotrapezoid nucleus/parafacial respiratory group Pre-Bötzinger complex Brainstem

a b s t r a c t In a previous study, we showed that in an in vitro en bloc preparation of newborn rats perfused with standard [K+ ] (6.2 mM) and high [K+ ] (11.2 mM) artificial cerebrospinal fluid (aCSF), nociceptin/orphanin FQ (N/OFQ) suppresses bursting of pre-inspiratory neurons with 1:1 coupling to the fictive inspiration. However, it is unclear whether the pre-Bötzinger complex (preBötC) is involved in the N/OFQ-induced slowing. Using in vitro en bloc preparations with and without the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) perfused with high [K+ ] aCSF, we found the following: (1) there were no differences in the effects of N/OFQ on the inspiratory rhythm between the preparations with and without the RTN/pFRG, (2) N/OFQ decreased the input resistance of inspiratory neurons (Insps) in the preparations without the RTN/pFRG and suppressed their ectopic firing activities, and (3) N/OFQ suppressed the spontaneous firing of Insps under a chemical synaptic transmission blockade. In conclusion, it is possible that the preBötC is involved in N/OFQ-induced inspiratory rhythm slowing. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the nociceptin/orphanin FQ peptide (NOP) receptor, a G proteincoupled receptor (Reinscheid et al., 1995). N/OFQ has been shown to act as a neuromodulator that slows the inspiratory rhythm in the medulla oblongata of in vitro brainstem-spinal cord preparations of newborn rat (Takita et al., 2003; Ruangkittisakul and Ballanyi, 2006), though the role of N/OFQ in breathing control in vivo remains unclear. It has been suggested that two distinct rhythmogenic networks in the rostral ventrolateral medulla (RVLM) are involved in primary respiratory rhythm generation. These include the preBötzinger complex (preBötC) in a limited region of the RVLM, in which inspiratory neurons (Insps) with endogenous bursting properties exist, and the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), which is mainly composed of pre-inspiratory neurons (Pre-Is) and surrounds the facial nucleus, although the exact location of the rhythmogenic component of the RTN/pFRG is not yet known (Smith et al., 1991; Onimaru et al., 1988, 2006; Onimaru and Homma, 2003; Mellen and Mishra, 2010; Mellen and Thoby-Brisson, 2012; Feldman et al., 2013). N/OFQ suppressed the bursting and spiking activity of PreIs with their 1:1 coupling to the fictive inspiration, causing

∗ Corresponding author. Tel.: +81 11 706 7861; fax: +81 11 706 7861. E-mail address: [email protected] (K. Takita). http://dx.doi.org/10.1016/j.resp.2014.12.005 1569-9048/© 2014 Elsevier B.V. All rights reserved.

non-quantal slowing of the inspiratory rhythm in an in vitro en bloc preparation (Takita and Morimoto, 2008). However, it is unclear whether the N/OFQ-induced non-quantal slowing is due to the selective suppression of the RTN/pFRG or whether the preBötC is also involved in the N/OFQ-mediated modulation of the respiratory rhythm. We previously attempted to elucidate the direct effects of N/OFQ on the preBötC using in vitro en bloc preparations with the RTN/pFRG perfused with elevated [K+ ] (11.2 mM) artificial cerebrospinal fluid (aCSF) (Takita and Morimoto, 2008), in which the preBötC is functionally uncoupled from the RTN/pFRG and generates an inspiratory rhythm without the excitatory drive from the RTN/pFRG (Mellen et al., 2003). We observed that the application of N/OFQ restored the functional coupling of the RTN/pFRG and the preBötC. This finding showed that after the application of N/OFQ, the RTN/pFRG is involved in respiratory rhythm generation even under conditions of high [K+ ]. Our previous study also showed that in vitro en bloc preparations perfused with high [K+ ] aCSF were less sensitive to N/OFQ than those perfused with lower [K+ ] (6.2 mM) aCSF (Takita and Morimoto, 2008). Accordingly, preparations in which the RTN/pFRG is removed should be investigated to elucidate the direct effects of N/OFQ on the preBötC. In addition, in vitro studies should be conducted in preparations without the RTN/pFRG and in those with the RTN/pFRG perfused with high [K+ ] aCSF to determine the effects of N/OFQ on the inspiratory rhythm generated by the preBötC alone as well as the effects of N/OFQ on the rhythm generated by coordination of the RTN/pFRG and the preBötC, respectively.

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The N/OFQ-induced slowing of the inspiratory rhythm results from the activation of NOP receptors in the medulla oblongata (Takita et al., 2003). Activation of the NOP receptor inhibits adenylyl cyclase via an inhibitory G protein (Reinscheid et al., 1995). The application of phosphodiesterase-4 inhibitors to elevate the intracellular cyclic AMP (cAMP) concentration has been shown to reverse the depressant action of N/OFQ on the inspiratory rhythm in in vitro en bloc preparations with the RTN/pFRG (Ruangkittisakul and Ballanyi, 2006). In brainstem slices without the RTN/pFRG, the methylxanthine caffeine reversed the inspiratory depression evoked by a ␮-opioid receptor agonist via phosphodiesterase-4 blockade, which resulted in elevated cellular cAMP levels (Ruangkittisakul and Ballanyi, 2010). However, it is unclear whether phosphodiesterase-4 inhibitors act on the RTN/pFRG or the preBötC to reverse the N/OFQ-induced slowing of the inspiratory rhythm. The aim of the present study was to elucidate whether the preBötC is involved in the N/OFQ-induced slowing of the inspiratory rhythm using in vitro newborn rat brainstem-spinal cord preparations with and without the RTN/pFRG perfused with high [K+ ] aCSF. We also examined whether the methylxanthine caffeine affects the action of N/OFQ on the preBötC. 2. Materials and methods 2.1. Standard in vitro en bloc preparations with the RTN/pFRG This study was approved by the Animal Care Committee of the Hokkaido University Graduate School of Medicine. Wistar rats (2–4 days old) were deeply anesthetized with sevoflurane until nociceptive reflexes induced by tail pinch were abolished, and the isolated brainstem and cervical spinal cord were obtained as described (Suzue, 1984). The brainstem was rostrally decerebrated between the VIth cranial nerve roots and the lower border of the trapezoid body. The preparation was placed in a small chamber (volume 1.5 ml) and continuously perfused at a rate of 3.0 to 5.0 ml/min with standard aCSF composed of 124 mM NaCl, 5.0 mM KCl, 1.2 mM KH2 PO4 , 2.4 mM CaCl2 , 1.3 mM MgSO4 , 26 mM NaHCO3 , and 30 mM glucose. The aCSF was equilibrated with 95% O2 and 5% CO2 to pH 7.4 at 27.5 ◦ C. Although the [K+ ] (6.2 mM) in standard aCSF is higher than physiological CSF [K+ ] (3 mM) for the long-term maintenance of respiratory network activity (Ruangkittisakul et al., 2007), the coupling of Pre-Is and the inspiratory cervical rhythm is preserved (Onimaru et al., 1988; Takita and Morimoto, 2008). The standard preparations were perfused with standard aCSF for more than 30 min, followed by perfusion with high [K+ ] aCSF (with 5 mM KCl added for a final concentration of 11.2 mM K+ ). 2.2. In vitro en bloc preparations without the RTN/pFRG (preBötC preparations) The RTN/pFRG was removed from a standard preparation as described (Takita and Morimoto, 2010). The standard preparation was isolated according to the method described above. This preparation was soaked in standard aCSF containing 2.5% agar at 40 ◦ C, and was right-angled between the brainstem and the spinal cord. This was immediately followed by immersion in ice, thus solidifying the agar. This agar-embedded brainstem-spinal cord preparation was mounted in the bath of a vibratome (DTK-1000, Dosaka EM, Kyoto, Japan) with the rostral end of the medulla facing upward and was bathed in standard aCSF. The brainstem was sectioned serially (150–200 ␮m thick) in the transverse plane until the facial nucleus disappeared and the landmarks (i.e., the nucleus ambiguus and inferior olive) were visible in the removed slice. The preBötC preparation was then transferred into a recording chamber

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and fixed by small pins with the cut surface of the rostral medulla facing up. This in vitro preparation without the RTN/pFRG was continuously perfused with high [K+ ] aCSF. 2.3. Electrophysiological recordings We monitored the respiratory activity corresponding to fictive inspiration at the C4 or C5 ventral root using suction electrodes. Recording signals were amplified and band-pass filtered (50 Hz to 3 kHz) (AB-621G, Nihon Kohden, Tokyo). The C4/5 activity was rectified and integrated with a time constant of 100 ms (EI-601G, Nihon Kohden). We made intracellular whole-cell recordings from Pre-Is and Insps using the blind patch clamp technique (Onimaru and Homma, 1992). A glass micropipette (GC100 TF, Harvard Apparatus, Edenbridge, Kent, UK) was pulled on a horizontal puller (Model P-87, Sutter Instruments, Novato, CA). The tip size was 1 to 1.5 ␮m (4–8 M). The electrodes were filled with a solution containing 130 mM potassium gluconate, 10 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, 0.3 mM Na-GTP, 1 mM CaCl2 , and 1 mM MgCl2 , with a pH of 7.2 adjusted using KOH. Intracellular electrodes were inserted into the RVLM in the standard preparations and the ventral respiratory column in the preBötC preparations. Positive pressure (30–40 mmHg) was applied to keep the electrode tip clean. Neurons were sought by advancing the electrode while monitoring amplified extracellular signals with a sound monitor. Respiration-related neurons were identified by their characteristic firing pattern and by their temporal correlation with the respiratory cycle of C4/C5 activity. Pre-Is firing usually occurred during the pre-inspiratory phase and the post-inspiratory phase under superfusion with standard aCSF. Discharge of Insps occurred during the inspiratory phase. When a target neuron was found, the positive pressure was released and negative pressure was applied. The resulting formation of a gigaohm (>1 G) seal was monitored and confirmed by applying a hyperpolarizing current pulse (0.1 nA; duration 30 ms). When this seal was established, the pressure was returned to zero. Rupture of the cell membrane was achieved by applying negative pressure (40–100 mmHg) often together with a single hyperpolarizing current pulse (1.0 nA; duration 30 ms). The membrane potential was recorded with a single electrode voltage clamp amplifier (CEZ-3100, Nihon Kohden) after compensation for the series resistance and capacitance. The input membrane resistances of neurons were determined according to the voltage changes in response to DC current pulses (500 ms, 40 pA). We recorded the extracellular unit activity of the respiratoryrelated neurons using the same glass microelectrodes as those used for the intracellular recordings. The C4/C5 and neuronal activities were monitored via an analog-digital converter and data acquisition software (MacLab, AD Instruments, Castle Hill, Australia). Data were sampled (sampling rate 0.4–4 kHz) and stored on a computer for off-line analysis. 2.4. Experimental protocols and drug application To examine the effects of N/OFQ on inspiratory rhythm and PreIs and Insps activity, we administered N/OFQ (Tocris Bioscience, Bristol, UK) for 15 min to standard or preBötC preparations through the recording chamber by means of a gravity-driven system. In the standard preparations, N/OFQ was applied 20 min after the standard aCSF was switched to control high [K+ ] aCSF. To further examine whether methylxanthines or a gamma-aminobutyric acid-A (GABAA ) receptor antagonist affected the action of N/OFQ, the administration of N/OFQ was followed by a co-application of N/OFQ and either caffeine (Wako Pure Chemical Industries, Osaka, Japan) or a GABAA receptor antagonist, (−)-bicuculline methiodide

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(Abcam, Cambridge, UK), for 15 min. Each preparation was exposed one time to a single concentration of N/OFQ. To block chemical synaptic transmission, the Mg2+ concentration of the high [K+ ] aCSF was increased to 5 mM and its Ca2+ concentration was reduced to 0.2 mM (low [Ca2+ ]/high [Mg2+ ] aCSF) (Onimaru et al., 1989; Johnson et al., 1994). The preBötC preparations were perfused with low [Ca2+ ]/high [Mg2+ ] aCSF for 40 min, and the ventral respiratory column was then explored with an extracellular recording electrode to locate spontaneously firing neurons. Experiments were carried out according to the following protocols. (1) To examine the direct effects of N/OFQ on Insps with intrinsic oscillatory properties, N/OFQ was applied under low [Ca2+ ]/high [Mg2+ ] conditions for 15 min, followed by the co-application of N/OFQ and caffeine for 15 min. The low [Ca2+ ]/high [Mg2+ ] aCSF was then switched to control high [K+ ] aCSF that included caffeine. (2) To examine the effects of N/OFQ on the intrinsic oscillatory Insps under the conditions in which the medullary respiratory rhythmogenic networks were intact, the low [Ca2+ ]/high [Mg2+ ] aCSF was switched to the control high [K+ ] aCSF and N/OFQ was applied for 15 min after the recovery of the inspiratory rhythm. 2.5. Data analysis We measured the C4 burst frequency and the inspiratory duration (Ti) of the integrated C4 discharge as well as the burst rates, number of action potentials, resting membrane potential, and input membrane resistance of the Pre-Is and Insps. We calculated the intraburst spike frequency (i.e., the number of intraburst action potentials/Ti) in the Insps. The average values were calculated from bursts recorded during a 2 to 3 min period. All data are presented as the means ± standard deviation (SD). A two-way factorial analysis of variance (ANOVA) was used to compare the concentration-response curves of N/OFQ in the standard and preBötC preparations. A Student’s paired t-test or one-way ANOVA for repeated measures with Bonferroni tests were performed to distinguish within-group differences over time. Repeated measures ANOVA followed Mauchly’s sphericity test. When the assumption of sphericity was violated, the degree of freedom was modified using the Greenhouse–Geisser epsilon. p-values