Site-specific hydrogen sulfide-mediated central regulation of respiratory rhythm in medullary slices of neonatal rats

Neuroscience 233 (2013) 118–126

SITE-SPECIFIC HYDROGEN SULFIDE-MEDIATED CENTRAL REGULATION OF RESPIRATORY RHYTHM IN MEDULLARY SLICES OF NEONATAL RATS L. CHEN, a J. ZHANG, b Y. DING, c H. LI, a L. NIE, d H. ZHOU, a Y. TANG a AND Y. ZHENG a*

INTRODUCTION Hydrogen sulfide (H2S) was the third gaseous signaling molecule to be discovered, following nitric oxide and carbon monoxide (Wang, 2002). It is endogenously produced and participates in a wide range of biological activities in mammals. H2S is produced by three enzymes, cystathionine beta-synthase (CBS), cystathionine gammalyase (CSE) and 3-mercaptopyruvate sulfotransferase (3MST; Abe and Kimura, 1996; Hosoki et al., 1997; Shibuya et al., 2009). The expression of these enzymes is tissue- and cell type-specific. CBS is expressed at high levels in the hippocampus and the cerebellum (Abe and Kimura, 1996), mainly in astrocytes (Enokido et al., 2005; Ichinohe et al., 2005). CSE is expressed mainly in the cardiovascular system (Hosoki et al., 1997) and 3MST is localized in neurons (Shibuya et al., 2009). In the central nervous system, H2S is generated mainly by CBS in astrocytes and 3MST in neurons. H2S facilitates induction of hippocampal long-term potentiation by enhancing the activity of NMDA receptors (Abe and Kimura, 1996). It may also participate in regulation of blood pressure and heart rate via interaction with ATPsensitive potassium channels (KATP channels) in the hypothalamus (Dawe et al., 2008). H2S protects neurons from oxidative stress by increasing glutathione production (Kimura and Kimura, 2004; Kimura et al., 2010) and opening potassium channels (Pan et al., 2010). H2S is involved in central regulation of respiratory rhythm. CBS has been found in the brain stem of rats (Abe and Kimura, 1996). Inhalation of exogenous H2S at moderate concentrations increases respiratory rate, but high concentrations decrease respiratory rate (Beauchamp et al., 1984; Reiffenstein et al., 1992). Our previous study (Hu et al., 2008) showed that a moderate concentration of NaHS (an H2S donor) (100–300 lM) induces a biphasic change in the rhythmic activity of the hypoglossal rootlets in 1200-lm thick medullary slices. Burst frequency (BF) showed an initial inhibition followed by an excitation. These effects were mimicked by the CBS substrate cysteine, and the biphasic response induced by cysteine was eliminated by application of the CBS inhibitor hydroxylamine (Hu et al., 2008). We also showed that, in thin (800–900 lm) slices, 200 lM NaHS induced the excitatory effect only, with no initial inhibition of BF (Pan et al., 2010). The caudal levels of the slices used in both studies were 100 lm caudal to the obex of the medulla oblongata. Therefore, the thicker (1200 lm)

a

Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, PR China

b

Department of Physiology, Chengdu Medical College, Chengdu, PR China

c Department of Histology and Embryology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, PR China d

Department of Physiology, Ningxia Medical University, Yinchuan, PR China

Abstract—Hydrogen sulfide (H2S) is involved in central regulation of respiratory rhythm at the level of the medulla oblongata. The present study was carried out to test our hypothesis that H2S exerts site-specific regulatory action on respiratory rhythm in the medulla oblongata of neonatal rats. The rhythmic discharge of hypoglossal rootlets in medullary slices of neonatal rats was recorded. 200 lM NaHS (an H2S donor) increased burst frequency (BF) in 900-lm slices containing the pre-Bo¨tzinger complex (preBo¨tC), whereas it caused diphasic responses in 1200-, 1400- and 1800-lm slices containing both the preBo¨tC and part or all of the parafacial respiratory group (pFRG): an initial decrease in BF followed by an increase. The initial decrease in BF was no longer observed after unilateral lesion of the pFRG region in the 1400-lm slices. In addition, BF was increased by a unilateral micro-injection of NaHS into the preBo¨tC region, but was decreased by an injection into the pFRG region. These data support our hypothesis that the regulatory action of H2S on respiratory rhythm in the medulla oblongata is site-specific. The excitatory effect is caused by the preBo¨tC, while the inhibitory effect is from the pFRG. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: hydrogen sulfide, respiratory rhythm, pre-Bo¨tzinger complex, parafacial respiratory group, medullary slice, neonatal rat.

*Corresponding author. Address: Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, PR China. Tel: +86-28-8550-2389; fax: +86-28-8550-3204. E-mail address: [email protected] (Y. Zheng). Abbreviations: 3MST, 3-mercaptopyruvate sulfotransferase; ACSF, artificial cerebrospinal fluid; BD, burst duration; BF, burst frequency; BI, burst interval; CBS, cystathionine beta-synthase; CSE, cystathionine gamma-lyase; H2S, hydrogen sulfide; IA, integrated amplitude; IO, inferior olivary nucleus; pFRG, parafacial respiratory group; preBo¨tC, pre-Bo¨tzinger complex; pre-I, pre-inspiratory.

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.12.047 118

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slices used in our previous study (Hu et al., 2008) would contain both the pre-Bo¨tzinger complex (preBo¨tC) and the caudal part of the parafacial respiratory group (pFRG; Smith et al., 1991; Onimaru and Homma, 2003; Hu et al., 2008), and the thinner (800–900 lm) slices used by Pan et al. would contain only the preBo¨tC (Smith et al., 1991). Both the preBo¨tC and pFRG have been shown to produce intrinsic periodic bursts in the medulla of rodents and are known as respiration-related rhythm generators. However, the preBo¨tC produces inspiratory bursts and the pFRG generates predominantly pre-inspiratory (pre-I) activity (Smith et al., 1991; Onimaru and Homma, 2003; Feldman and Del Negro, 2006). We hypothesized that these two regions may play different roles in H2S-mediated effects on the respiratory-like rhythmic activity, i.e., that sitespecificity exists in the regulatory actions of H2S on respiration.

EXPERIMENTAL PROCEDURES Animals and reagents Experiments were performed on medullary slices of male or female neonatal (P0–3) Sprague–Dawley rats. All procedures were reviewed and approved by the Sichuan University Committee on the Use of Live Animals in Research, which is in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. NaHS and Pontamine Sky Blue were purchased from Sigma–Aldrich (St. Louis, MO, USA).

Preparation of medullary slices Medullary slices were prepared as described previously (Pan et al., 2010). In brief, the animals were anesthetized with ether by inhalation and then decapitated. Brainstems were isolated in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 3 KCl, 2 CaCl2, 1 MgSO4, 22 NaHCO3, 1 KH2PO4 and 30 D-glucose, equilibrated with carbogen (95% O2 and 5% CO2), pH 7.4, and then glued, caudal-end upwards, against a cube of agar. Specimens were serially sectioned to obtain medullary slices with different thicknesses: 900, 1200, 1400 and 1800 lm; starting from 100 lm caudal to the obex, up to the corresponding rostral levels of the medulla oblongata and retaining the hypoglossal rootlets. 900-lm slices contained the preBo¨tC (Smith et al., 1991), 1200-lm slices contained the preBo¨tC and the caudal part of the pFRG (Onimaru and Homma, 2003; Hu et al., 2008), and the 1400-lm and 1800lm slices contained more or almost all of the pFRG, respectively. Fig. 1 shows five representative coronal planes and one sagittal plane of the medulla oblongata of a neonatal rat, to indicate the location of the cuts relative to the structures of interest.

Recording of hypoglossal rootlets discharge Medullary slices were continuously perfused with ACSF bubbled with carbogen at a rate of 4 ml/min at 27–28 °C, pH 7.4, and the discharge from the hypoglossal rootlets was recorded. To maintain consistent burst activity, the KCl concentration of the perfusing ACSF was raised from 3 to 8 mM. Slices were incubated in this solution for 30 min before beginning experiments. Glass suction electrodes filled with ACSF were

Fig. 1. Photographs of different planes of medulla oblongata of neonatal rat with HE staining. (A) Coronal planes. (B) Sagittal plane. The solid vertical lines indicate the levels of cuts made to obtain medullary slices of different thicknesses. Abbreviations: Bo¨tC, Bo¨tzinger complex; cVRG, caudal ventral respiratory group; IO, inferior olivary nucleus; NA, ambiguous nucleus; NAc, compact ambiguous nucleus; NTS, nucleus tractus solitarius; pFRG, parafacial respiratory group; preBo¨tC, pre-Bo¨tzinger complex; py, pyramidal tract; rVRG, rostral ventral respiratory group; SP5, spinal trigeminal nucleus; XII, hypoglossal nucleus. Scale bars = 500 lm.

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used to record the discharge from the cut ends of the hypoglossal rootlets. Signals were amplified, filtered (s = 0.001 s, F = 1 kHz) and integrated (with a time constant of 50 ms), using the BL-420F biological signal processing system (Taimeng Biotech. Co., China). The burst duration (BD), burst interval (BI), BF and integrated amplitude (IA) of the activity of hypoglossal rootlets in the slices were analyzed.

Perfusion of slices with NaHS The slices were divided into four groups of different thicknesses: 900 lm (n = 6), 1200 lm (n = 7), 1400 lm (n = 6) and 1800 lm (n = 6). The hypoglossal discharge was recorded for 4 min in ACSF as baseline. Slices were then perfused with 200 lM NaHS for 10 min, followed by washout with ACSF for 10 min.

Nuclei micro-injection The effects of unilateral micro-injection (50 nl) of 200 lM NaHS or ACSF into the preBo¨tC, pFRG, hypoglossal nucleus (XII) or inferior olivary nucleus (IO) on the rhythmic discharge were examined in 1400-lm slices. The preBo¨tC is located about 300–400 lm rostral to the obex in the neonatal rat (Smith et al., 1991). Slices were placed into the recording chamber with caudal ends upward, and glass capillaries with 30–40lm tip diameter containing drugs were inserted 400–450 lm vertically into the ventral lateral medulla, to reach the preBo¨tC. The pFRG is located ventral, lateral and caudal to the facial nucleus (VII), between 800 lm rostral and 200 lm caudal to the most caudal level of the VII in neonatal rats (Onimaru and Homma, 2003; Ballanyi et al., 2009). Slices were placed into the recording chamber rostral end upward, and the glass capillary was inserted about 250 lm vertically into the VII region to reach the caudal level of the pFRG. The XII is located in the dorsomedial part of the medulla, beneath the base of the fourth ventricle. Slices were placed into the recording chamber caudal cut-surface upward, and the glass capillary was inserted 300–400 lm vertically into the dorsal medulla to reach the XII. The IO is located in the ventral part of the medulla. Slices were placed into the recording chamber caudal end upward, and the glass capillary was inserted 300–400 lm vertically into the ventral medulla to reach the IO. Slices were randomly divided into six groups, containing different nuclei and injected with either NaHS or ACSF: preBo¨tC-ACSF (n = 8), preBo¨tC-NaHS (n = 7), pFRG-ACSF (n = 7), pFRG-NaHS (n = 7), XII-NaHS (n = 6) and IO-NaHS (n = 7). BD, BI, BF and IA of hypoglossal bursts were analyzed. All the glass capillaries for the nuclei micro-injections contained 1% Pontamine Sky Blue. After the electrophysiological observations, the specimens were serially sectioned into 100-lm slices to localize the position and diffusion extent of the drug, according to the presence of Pontamine Sky Blue.

Statistical analysis All the electrophysiological data at each time point were compared with the baseline before chemicals were applied. Normalized BD, BI, BF and IA of hypoglossal activity were reported as means ± SEM and statistically analyzed with repeated measure ANOVA followed by LSD test. Group comparisons were analyzed with paired Student’s t-test. P values <0.05 were considered statistically significant.

RESULTS Effects of H2S on hypoglossal discharge in slices of different thicknesses Analysis of the integrated signals from the discharge revealed that 200 lM NaHS shortened BI and increased BF in 900-lm slices from the fourth to tenth minute (P < 0.05, n = 6). The BI was decreased by 42.58% and the BF was increased by 77.32% at the eighth minute (P < 0.05, n = 6; Figs. 2 and 3A). NaHS did not significantly change BD or IA (P > 0.05, n = 6). In the 1200-lm slices (containing the preBo¨tC and caudal part of the pFRG), 200 lM NaHS induced a diphasic response of BI and BF: a short-term increase in BI and decrease in BF (initial 2 min), followed by a decrease in BI and an increase in BF from the fourth to tenth minute. BI was increased by 8.51% at the second minute and decreased by 38.96% at the eighth minute (P < 0.05, n = 7); BF was decreased by 6.00% at the second minute and increased by 66.40% at the eighth minute (P < 0.05, n = 7; Figs. 2 and 3B). In addition, BD increased by 12.71% at only the fourth minute (P < 0.05, n = 7) with no change in IA (P > 0.05, n = 7; Figs. 2 and 3B). In the 1400-lm slices (containing the preBo¨tC and the majority of the pFRG), NaHS also induced a diphasic response in BF. BF was decreased by 9.25% at the second minute and increased by 13.81% at the eighth minute. BI decreased by 14.54% at the eighth minute (P < 0.05, n = 6; Figs. 2 and 3C). BD was prolonged from the fourth to tenth minute (P < 0.05, n = 6), and by 21.81% at the tenth minute (P < 0.05, n = 6). IA did not change significantly (P > 0.05, n = 6; Figs. 2 and 3C). In the 1800-lm slices (containing the preBo¨tC and almost the entire pFRG), NaHS caused similar responses to those

Nuclei micro-injury Electrical micro-lesion was performed in the pFRG/VII region of 1400-lm slices through a glass electrode with 30–40-lm tip diameter containing enameled wire and ACSF, by applying a 100 ms, 50 lA pulse. The extent of the lesions was verified histologically in 50-lm transverse Nissl-stained sections. After unilateral electrical lesion of the pFRG/VII region, slices were perfused with 200 lM NaHS for 10 min followed by washout with ACSF for 10 min. The effects of NaHS on respiratory activity were compared between the pFRG intact group (control group; n = 6) and the lesion group (n = 7).

Fig. 2. Effects of NaHS on the integrated activity of rhythmic respiratory-like discharge of hypoglossal rootlets of medullary slices of neonatal rats in groups of 900-, 1200-, 1400- and 1800-lm thickness.

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Fig. 3. Effects of NaHS on hypoglossal discharge in slices with different thicknesses. The burst duration (BD), burst interval (BI), burst frequency (BF) and integrated amplitude (IA) were normalized to baseline (average value during 4 min before application of NaHS). A: 900-lm group, B: 1200lm group, C: 1400-lm group, D: 1800-lm group. ⁄P < 0.05, #P < 0.01 versus baseline.

observed in the 1400-lm slices, except for a 15.51% increase in IA at the eighth minute (P < 0.05, n = 6; Figs. 2 and 3D). BI was increased by 15.61% at the second minute (P < 0.05, n = 6) and decreased by 20.15% at the tenth minute (P < 0.05, n = 6). BF was decreased by 11.72% at the second minute and increased by 22.66% at the tenth minute (P < 0.05, n = 6). BD increased by 30.89% at the eighth minute (P < 0.05, n = 6; Figs. 2 and 3D). The hypoglossal activity in all groups recovered following washout with ACSF for 10 min (Figs. 2 and 3). Effects of micro-injection of NaHS into the medullary nuclei We examined the effects of micro-injection of 200 lM NaHS or ACSF into the preBo¨tC, pFRG, XII or IO on the hypoglossal discharge in 1400-lm slices. The discharge was not significantly affected by inserting the glass capillary into the preBo¨tC or pFRG (P > 0.05; data not shown) or by micro-injection of ACSF (50 nl) containing 1% Pontamine Sky Blue (P > 0.05, n = 8; Fig. 4A, B). However, micro-injection of 200 lM NaHS

into the preBo¨tC shortened BD by 8.13% and BI by 25.16% (P < 0.05, n = 7) and increased BF by 32.44% (P < 0.05, n = 7), with no effect on IA (Fig. 4A). Injection of NaHS into the pFRG, however, prolonged BD by 14.29% and BI by 26.05% (P < 0.05, n = 7), and decreased BF by 18.34% (P < 0.05, n = 7), with no effect on IA (P > 0.05, n = 7; Fig. 4B). Microinjection of NaHS into either XII (n = 6) or IO (n = 7) had no effect on BD, BI, BF or IA (P > 0.05; Fig. 4C, D). The sites of micro-injection and the diffusion extent of the drug in the coronal plane are shown in Fig. 5. Based on the diffusion extent of Pontamine Sky Blue in the coronal plane and the number of coronal slices dyed by the Pontamine Sky Blue, we estimate that the diffusion extent of the drug is approximately 500 lm on the rostrocaudal axis. Effects of NaHS on hypoglossal discharge after pFRG lesion After unilateral electrical lesion of the pFRG region in the 1400-lm slices, the discharge of hypoglossal rootlets did not change significantly. BD was 0.72 ± 0.04 s and

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Fig. 4. Effects of micro-injection of NaHS or ACSF into the preBo¨tC, pFRG, XII and IO on hypoglossal discharge. The burst duration (BD), burst interval (BI), burst frequency (BF) and integrated amplitude (IA) were normalized to baseline (average value during 4 min before application of NaHS). A: preBo¨tC group, B: pFRG group, C: XII group, D: IO group. ⁄P < 0.05, #P < 0.01 versus baseline.

0.74 ± 0.04 s, BI was 9.58 ± 0.75 s and 9.19 ± 0.58 s, BF was 6.04 ± 0.51 bursts/min and 6.18 ± 0.39 bursts/ min, and IA was 18.58 ± 4.75 lV s and 18.39 ± 4.98 lV s, in control and after lesion, respectively (P > 0.05, n = 7). Perfusion of 200 lM NaHS decreased BI (P < 0.05, n = 7) and increased BF (P < 0.05, n = 7) from the second minute to tenth minute in the slices with lesion of the pFRG region, responses similar to those observed in the 900-lm slices, whereas IA decreased (P < 0.05, n = 7) and there was no significant effect on BD (P > 0.05, n = 7; Fig. 6A). After unilateral lesion of the pFRG region, perfusion of 200 lM NaHS decreased BI by 37.01% and increased BF by 56.23% at the eighth minute (P < 0.05, n = 7), and decreased IA by 8.20% at the second minute and 11.70% at the fourth minute (P < 0.05, n = 7; Fig. 6A). However, in the control group of intact 1400-lm slices, 200 lM NaHS induced diphasic responses: a decrease in BF in the initial stage followed by an increase in the following stage (the same responses as mentioned previously). Compared with the control group (n = 6), BF was increased from the fourth to eighth minute (P < 0.05), and BD was decreased

from the fourth to tenth minute (P < 0.05) in the pFRG lesion group (n = 7; Fig. 6A). Fig. 6B shows the lesioned VII/pFRG region with Nissl staining.

DISCUSSION In in vitro medullary slices of neonatal rats, we found that NaHS could excite the hypoglossal rhythmic discharge of 900-lm medullary slices, and affect the discharge of 1200-, 1400- and 1800-lm slices in a diphasic mode: an initial decrease followed by an increase in the discharge frequency. These results indicate that regulation of respiratory rhythm by H2S varies by location. This conclusion was further supported by the observation that micro-injection of NaHS into the preBo¨tC region produced an excitatory effect, whereas micro-injection of NaHS into the pFRG region caused an inhibitory effect, and the initial inhibitory effect disappeared after unilateral lesion of the pFRG region. Endogenous H2S has been shown to participate in central regulation of respiratory rhythm at the level of the medulla oblongata (Hu et al., 2008; Pan et al., 2010). In the present experiments, we observed that

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Fig. 5. Photographs of the location and extent of Pontamine Sky Blue in slices after micro-injection into the medullary nuclei. A: preBo¨tC; B: pFRG; C: XII; D: IO. Abbreviations: IO, inferior olivary nucleus; NA, ambiguous nucleus; NTS, nucleus tractus solitarius; pFRG, parafacial respiratory group; preBo¨tC, pre-Bo¨tzinger complex; py, pyramidal tract; SP5, spinal trigeminal nucleus; XII, hypoglossal nucleus.

H2S increased the BF of hypoglossal rootlets in 900-lm slices containing the preBo¨tC, ambiguous nucleus and solitary nucleus, similar to the data of Pan et al. (2010). In addition to the aforementioned nuclei, the thicker (1200–1800 lm) medullary slices in the present experiment also contained the caudal part or the entirety of the pFRG (Onimaru and Homma, 2003; Hu et al., 2008), which are a group of rhythmogenic preinspiratory interneurons implicated in generation of respiratory rhythm (Onimaru and Homma, 2003). H2S affected the discharge of these thicker slices in a diphasic mode, accompanied by a prolongation of inspiratory duration, a result which is similar to the data of Hu et al. (2008). H2S also increased the IA of the discharge in 1800-lm slices. These data indicate that the regulation of respiratory rhythm in the medulla oblongata by H2S varies depending on location. Our results suggest that the excitatory effect of H2S on respiratory rhythm might be caused by acting on the caudal medulla containing the preBo¨tC, and its inhibitory effect on BF and prolongation of inspiratory duration might be induced by acting on the more rostral part of the medulla containing the pFRG. The increase in IA induced by H2S might be caused by acting on the most rostral 400 lm of the 1800-lm slices. However, the precise locus or loci of action of H2S on IA remains to be determined. The preBo¨tC in the ventrolateral medulla is hypothesized to be a kernel for the generation of respiratory rhythm in vitro and in vivo (Smith et al., 1991; Gray et al., 2001; Tan et al., 2008). Serial transections of neonatal rat medulla eliminate respiratory rhythm when the preBo¨tC is abolished (Smith et al., 1991). Toxininduced slow neurodegeneration of neurokinin 1

receptor-expressing preBo¨tC neurons leads to a severe ataxic rhythm during wakefulness and apnea during sleep in adult rats (Gray et al., 2001; McKay et al., 2005). Rapid silencing of somatostatin-expressing neurons in the preBo¨tC in awake rats induces a persistent apnea without any respiratory movement (Tan et al., 2008). In neonatal rats, the preBo¨tC is 500 lm caudal to the most caudal level of VII and 300–400 lm rostral to the obex (Smith et al., 1991; Onimaru and Homma, 2003; Ballanyi et al., 2009). H2S induced an excitatory effect on the respiratory rhythm in 900-lm medullary slices, which contain the preBo¨tC. We speculated therefore that the preBo¨tC might be one of the sites of action of H2S. In the present experiment, microinjection of H2S into the preBo¨tC region increased BF by 32.44%. However, the peak change in BF induced by 10-min perfusion of NaHS in the 900-lm slices was 77.32%, which was greater than that induced by micro-injection. There are multiple possible reasons for this discrepancy: (1) in the perfusion experiment, H2S could act on the whole respiratory neural network in the 900-lm slices (including bilateral preBo¨tC), and in the micro-injection experiment, the drug action was confined to the unilateral preBo¨tC region. (2) In the perfusion experiment with 900-lm slices, the excitatory effect on respiration increased gradually with time, reaching a peak at the eighth minute, indicating a time-dependent effect; however, the micro-injection was a one-off process, so the drug action was transient. (3) The possibility that H2S may also act on other respiratory-related nuclei, such as the gigantocellular reticular nucleus, lateral paragigantocellular nucleus and nucleus raphe obscurus, could not be ruled out. The pFRG, composed predominantly of pre-I neurons as well as some inspiratory and expiratory neurons that

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Fig. 6. Effects of NaHS on hypoglossal discharge after electrical lesion of the pFRG region. (A) Changes in burst duration (BD), burst interval (BI), burst frequency (BF) and integrated amplitude (IA) of activity in 1400-lm slices induced by NaHS after unilateral damage to pFRG. ⁄P < 0.05, ⁄⁄ P < 0.01 versus baseline; #P < 0.05, ##P < 0.01 pFRG intact group (control) versus pFRG lesion group (lesion). (B) Photograph of a medullary slice of neonatal rat with Nissl staining. The higher magnifications of the boxed areas are shown in the lower panels. The lesioned VII/pFRG region is on the left and the control on the right.

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are involved in respiratory rhythm generation, was identified initially by optical recordings of respiratory neuron activity in the rostral ventrolateral medulla of neonatal rats (Onimaru and Homma, 2003). The pFRG might also control the activity of motor neurons innervating the abdominal expiratory muscles, and thus is considered to be an expiratory rhythm generator (Janczewski et al., 2002; Janczewski and Feldman, 2006). In the present experiment, H2S induced an initial decrease followed by an increase in BF, accompanied by a prolongation of BD in the thicker slices containing the pFRG, whereas it only increased the BF in the thin (900 lm) slices, which may not contain the pFRG. Micro-injection of NaHS into the pFRG/VII region decreased BF and prolonged BD. In addition, after unilateral lesion of the pFRG/VII region, the NaHS-induced inhibitory effect on BF in the initial stage and the prolongation of BD were eliminated. These data suggest that the inhibitory effect on BF and the prolongation of BD induced by H2S might result from its action on the pFRG/VII region. H2S might have different effects on respiratory rhythm depending on whether it is acting on the pFRG/VII region or the preBo¨tC region. The pFRG is located ventral, lateral and caudal to the VII, and some pre-I neurons are distributed on the edge of the VII; the two nuclei are very close (Onimaru and Homma, 2003). In our micro-injection and lesion experiments, to ensure the whole pFRG was reached, the VII was also inevitably affected. The VII is mainly a motor nucleus, involved in regulation of facial activity such as expression, chewing, swallowing and sucking. Some branches of the facial nerve have rhythmic respiratory-like discharge activity, and may innervate the alae nasi and play a role in reducing nasal airway resistance in the process of breathing (Strohl, 1985; Hwang et al., 1988). The activity of pre-I neurons in the pFRG occurs in phase with the facial nerve output in the rostral medulla, and the facial nerve activity is presumably derived from the pre-I neurons, since rhythmic activity of the facial nerve disappears when the burst activity of putative pre-I neurons is stopped (Onimaru et al., 2006). Furthermore, the dorsal and lateral facial subnuclei have been shown to be respiratory-modulated (Persson and Rekling, 2011). Thus, we believe that the regulatory effect of H2S on the respiratory rhythm in the medullary slices by acting on the pFRG/VII region may result mainly from the pFRG. However, interneurons of VII would project to the nucleus tractus solitarius, nucleus ambiguous and ventrolateral reticular formation in the medulla oblongata, suggesting that the facial nucleus may be involved in the regulation of respiratory activity (Li et al., 2004). Therefore, in the present experiments we could not rule out the possibility that H2S may have also been acting on the VII to affect respiration. There are at least two respiration-related rhythm generators in the medulla oblongata, which interact with each other as a coupled oscillator to regulate breathing (Mellen et al., 2003; Feldman and Del Negro, 2006). The preBo¨tC produces inspiratory neuron bursts (Smith et al., 1991), and the pFRG produces predominantly

pre-I neuron bursts (Onimaru and Homma, 2003). These two rhythm generators respond differently to modulatory inputs, including lung stretch receptor afferents (Mellen and Feldman, 2001), as well as some chemical signals, such as opiates, serotonin (Onimaru et al., 1998) and changes in extracellular K+ concentration. Opiates slow down the inspiratory neuron activity and the bursting frequency of the preBo¨tC (Gray et al., 1999; Onimaru et al., 2006), but have no effect on pre-I neuron activity (Takeda et al., 2001). High K+ concentration can increase the burst frequency of the preBo¨tC (Smith et al., 1991), but disturbs the activity of pre-I neurons (Mellen et al., 2003; Onimaru et al., 2006). This differential sensitivity of the two respiration oscillators extends the robustness of respiratory rhythm generation beyond that of either constituent oscillator (Mellen et al., 2003). Our research shows that H2S, a very important gaseous signaling molecule, also regulates the respiratory rhythm by acting differently on the two respiration-related rhythm generators. The effects of H2S on the respiratory activity might be mediated by opening KATP channels in the initial inhibitory stage and activating the adenylate cyclase– cAMP pathway in the later excitatory stage (Hu et al., 2008). However, the precise neuronal mechanisms of actions of H2S on the rhythmic respiratory activity of the medullary slices remain to be investigated.

CONCLUSION H2S exerts central regulation of respiratory rhythm via a site-specific mechanism of action. Its excitatory effect is mediated by the preBo¨tC, and its inhibitory effect by the pFRG. Acknowledgments—This work was supported by grants from the National Natural Science Foundation of China (Nos. 30971073 and 31271233) and the Fund of Doctoral Program of Ministry of Education of China (No. 20100181110048). The authors thank Dr. Jerry Yu for his critical scientific comments and editorial review.

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(Accepted 17 December 2012) (Available online 3 January 2013)