cGMP signalling pathways

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Modulation of synaptic and channel activities in the respiratory network of the mice by NO/cGMP signalling pathways Sergej L. Mironov⁎, Kersten Langohr DFG-Center of Molecular Physiology of the Brain, Department of Neuro- and Sensory Physiology, Georg-August-University, Göttingen, Humboldtallee 23, 37073, Germany

A R T I C LE I N FO

AB S T R A C T

Article history:

We examined signalling pathways which can involve NO as a second messenger in the

Accepted 17 September 2006

respiratory network. In the functional slice preparation, NO donors depressed the

Available online 13 December 2006

respiratory motor output and enhanced its depression after brief episodes of hypoxia. In the inspiratory neurons, NO donors suppressed spontaneous excitatory and inhibitory

Keywords:

synaptic currents, activated single KATP channels and inhibited L-type Ca2+ channels. NO

Nitric oxide

scavengers, PTIO and hemoglobin, and the blocker of NO synthase, N-monomethyl-L-

Respiratory network

arginine, induced effects opposite to those of NO donors and indicated the role of

Synaptic current

endogenously generated NO in the modulation of the respiratory activity. Using

K(ATP) channel

fluorescent dyes DAF-2 and DCF, we imaged NO and reactive oxygen species (ROS).

Ca(L) channel

Concentrations of NO and ROS increased during brief episodes of hypoxia and they both

Hypoxia

contributed to the activation of KATP channels due to oxygen withdrawal. The oxidizing agent t-butyl-hydroperoxide acted similarly to NO donors but it did not interfere with the effects of NO. Increase in cGMP levels with 8-Br-cGMP reproduced the actions of NO donors and occluded the effects of their subsequent applications. We propose that in the respiratory neurons, a constitutive production of NO is responsible for a tonic activation of cGMP-coupled signalling pathways and changes in NO levels modulate the respiratory motor output by altering the activity of KATP and L-type Ca2+ channels. © 2006 Published by Elsevier B.V.

1.

Introduction

Nitric oxide, a membrane-permeant messenger in various physiological and pathological processes, acts as a local neuromodulator in the CNS (cf. Prast and Philippu, 2001;

Keynes and Garthwaite, 2004; Calabresi et al., 2000). NO modifies electrical activity via NO/cGMP pathway and reversible oxidization of sulfhydryl (–SH) groups of corresponding channels and receptors (Jacintho and Kovacic, 2003; Fischmeister et al., 2005; Xu et al., 2004). NO synthases (NOS) are

⁎ Corresponding author. Fax: +49 551 39 5917. E-mail address: [email protected] (S.L. Mironov). Abbreviations: CaL, L-type Ca2+ channels; DCF, carboxy-H2CFDA;GC, guanylyl cyclase; IR-DIC, infrared differential interference contrast; KATP, ATP-sensitive K+ channels; NMMA, N-monomethyl-L-arginine; NOS, NO synthase; NTS, nucleus tractus solitarii; ODQ, guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one; ROS, reactive oxygen species; SNAP, S-nitroso-N-acetylpenicillamine; tBuHQ, t-butyl-hydroperoxide; PKG, cGMP-activated proteinkinase; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; TMRE, tetramethylrhodamineethylester 0006-8993/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.brainres.2006.09.114

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responsible for constitutive production of NO and it can be additionally enhanced during ischemia and hypoxia due to disturbances in energy metabolism (Bolanos and Almeida, 1999; Keynes and Garthwaite, 2004). In the heart, several ion channels have been shown to be modified by NO, such as L-type Ca2+ (CaL), ATP-sensitive (KATP), and pacemaker f-channels (Fischmeister et al., 2005) that is mediated by cGMP. KATP channels and NOS activity are important in cardioprotection (Xu et al., 2004) and neuroprotection (Bolanos and Almeida, 1999; Moncada and Bolanos, 2006) that also involves the activation of NO-cGMP-PKG signalling pathway. Whole-animal studies have shown that NO markedly affects sympathetic outflow, alter respiratory rhythm and influence pain thresholds in the spinal cord. The activation of the NO/cGMP pathway causes direct blockade of acute and persistent hypernociception by opening KATP channels via stimulation of PKG (Sachs et al., 2004). Sensory neurons respond to hypoxia with an activation of NOS due to Ca2+ influx via CaL channels that results in enhanced NO production which is associated with mitochondria and contributes to resistance against hypoxia (Henrich et al., 2004). Nitric oxide acts as a retrograde messenger in the nucleus tractus solitarii (NTS) (Ogawa et al., 1995) where a reciprocal regulation of NO and glutamate exists (Lin et al., 2000). If similar signalling pathways are expressed in the respiratory network which is located ventral to NTS, this would modulate the respiratory motor output driven by glutamatergic interneurons which are also involved in the actions of hypoxia on breathing (Kline et al., 1998). In the rat and the cat brainstem, chronic pretreatment of rats with NOS blockers causes a significant decrease in cGMP, and attenuates the ventilatory response to hypoxia (Haxhiu et al., 1995) which is consistent with enhanced NO production during hypoxia as observed in other brain regions (Bolanos and Almeida, 1999; Keynes and Garthwaite, 2004). NO actions in respiratory neurons have not been yet investigated. We here report that NO donors and NOS inhibitors modulated the respiratory motor output in the functional slice preparation. The two classes of drugs produced opposite effects. The modulation of the respiration-related variables was accompanied by changes in the amplitude but not in the frequency of spontaneous synaptic currents that indicated postsynaptic mechanisms of NO actions. In the presence of NO donors and NOS inhibitors, the response of the respiratory network to hypoxia was also modified that involved modulation of the activity of KATP and CaL channels in the inspiratory neurons. Using DAF-2 and carboxy-H2CFDA (DCF), the dyes which respectively sense NO and reactive oxygen species (ROS), we observed a localized production of both moieties in the vicinity of mitochondria which was enhanced during hypoxia. The actions of NO on synaptic and ion channel activities did not interfere with the effects of ROS. Instead, all observed responses which involved NO were reproduced by elevations of intracellular cGMP levels. Thus, regulation of respiratory rhythmogenesis by NO/cGMP complements previously described mechanisms of respiratory rhythm modulation utilizing protein kinases A and C, and G-proteins (Haji et al., 1996; Lalley et al., 1997; Johnson et al., 1996; Pierrefiche et al., 1996; Mironov and Richter, 2000a,b).

2.

Results

In order to reveal the mechanisms of NO actions in the respiratory network we used specific agents which act on different steps in the signalling pathways involving NO. Fig. 1 represents their effects on the respiratory motor output. The actions of NO donors and NOS inhibitors on the respirationrelated variables, synaptic currents and the activity of CaL and KATP channels are summarized in Table 1. 300 μM SNAP (NO donor) decreased the amplitude and the frequency of the respiratory motor output (Fig. 1) that developed in parallel with suppression of the synaptic drives in the inspiratory neurons. Other NO donors tested, sodium nitroprusside (300 μM), and photochemically activated KRu(NO)Cl 5 (100 μM), acted similarly and the differences from the effects of SNAP were not significant. Application of NOS inhibitor, LNMMA enhanced the respiratory rhythm that probably reflected constitutive generation of endogenous NO in the tissue. NO scavengers, PTIO, N-acetyl-cysteine (1 mM) and hemoglobin (10 μM), evoked the effects which similar to LNMMA, and in their presence the actions of SNAP were not observed. NO is free radical and it can act as reactive oxygen species, therefore we next examined whether NO effects could be mimicked by oxidative agents. t-BuHQ depressed the rhythm but this did not prevent a subsequent depression by SNAP (Fig. 1). Other redox agents such as N-ethylmaleimide and dithiotreitol (both applied at 0.1 mM) which respectively oxidize and reduce sulfhydryl groups, correspondingly depressed and enhanced the respiratory motor output (n = 4 for both), which however did not modify the effects of NO donors. The membrane-permeable cGMP analogue, 8-Br-cGMP (300 μM), depressed the respiration-related variables and subsequent application of SNAP was ineffective (Fig. 1). Inhibitor of cGMP phosphodiesterase zaprinast (20 μM) produced similar effects (n = 4). We also examined whether the effects of NO donors were blocked by ODQ, a potent and selective inhibitor of guanylyl cyclase. Preincubation of slice with this GC inhibitor (50 μM for 30 min) abolished NOmediated suppression of the rhythm (n = 5), indicating that NO effects on the respiratory motor output were primarily mediated by GC-dependent pathway. The respiratory motor output is generated within a neuronal network and is established via synaptic interactions (cf. Richter and Spyer; Feldman et al., 2003). The actions of NO donors and NOS inhibitors on spontaneous excitatory and inhibitory synaptic currents are summarized in Table 1. The main effect involved changes in the amplitude of synaptic currents and the frequency was not modified which indicated a postsynaptic target of NO in the inspiratory neurons. Inhibitory and excitatory synaptic currents decayed according to mean time-constants 3.9 ± 0.5 ms and 3.5 ± 0.4 ms, which increased in the presence of SNAP (300 μM) to 5.1 ± 0.6 ms and 4.6 ± 0.5 ms (n = 5, p = 0.1), respectively. Because NO activated KATP channels (see below), we tested their activator, diazoxide (100 μM). In its presence the time-constants of IPSC and EPSC increased to 4.9 ± 0.6 ms and 4.5 ± 0.5 ms, respectively (n = 5) and did not change further after addition of SNAP. According to Eq. (2) the observed increases in time-constants should

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Fig. 1 – Modulation of respiration-related activities by NO. A—Each panel shows two traces: the respiratory motor output which was recorded from the hypoglossal (XII) nerve and the whole-cell current in the inspiratory neurons (Im) which was recorded at −40 mV. NO donor SNAP (300 μM), NOS inhibitor L-NMMA (500 μM), oxidizing agent t-BuHQ (100 μM), and membrane permeable analogue 8-Br-cGMP (300 μM) were applied as indicated by horizontal bars. In the lowermost panel, SNAP was added after preincubation of the slice with the inhibitor of guanylyl cyclase (ODQ, 50 μM, 30 min). B—Synaptic currents measured before and 5 min after beginning the drug applications. The inhibitory and excitatory synaptic currents correspond to the brief upward and downward-directed transients, respectively, and synaptic drives are the longer lasting current decreases, which are in phase with the inspiratory motor output. Note a depression of the respiration-related activities by SNAP in the presence of t-BuHQ and the absence of SNAP effects after pretreatment of slices with Br-cGMP and ODQ.

decrease the amplitude of synaptic currents by about 1.5-fold which is in line with the data given in Table 1. Beyond the effects on the rhythm, NO modulated the response of the respiratory network to hypoxia (Fig. 2). The reaction consists of an early augmentation of the respiratory output which is followed by depression (Mironov and Richter, 2000a,b, 2001; Mironov and Langohr, 2005; Mironov et al., 1999). In the presence of NO donors, the duration of augmentation was shorter and subsequent depression lasted longer (Fig. 2B, 39 ± 5 vs. 26 ± 4 s and 65 ± 7 vs. 93 ± 8 s, respectively, n = 9, p = 0.03). NOS inhibitors produced opposite effects and mean durations of augmentation and depression correspondingly changed to 52 ± 7 and 48 ± 4 s (Fig. 2C, n = 9, p = 0.05). L-type Ca2+ channels (Mironov and Richter, 2000b; Mironov and Langohr, 2005) and KATP channels (Mironov et al., 1998,

1999) modulate the respiratory activity and are respectively responsible for its augmentation and depression during hypoxia. NO donors activated KATP channels (Fig. 3). Singlechannel activities were monitored continuously in the cellattached patches and the openings at a conductance level around 75 pS, appeared as inward deflections (Fig. 3). KATP channels showed gating pattern and sensitivity to hypoxia, diazoxide and suphonylureas as described previously (Mironov et al., 1998, 1999). The activity of KATP channels slowly increased after initiating photochemical production of NO from photolabile NO donor KRu(NO)Cl5 (Fig. 3A). In the presence of NO scavenger PTIO (0.1 mM), the activity of channels decreased (Fig. 3B). Oxidizing agent t-BuHQ activated KATP channels (popen increased from 0.12 ± 0.02 to 0.28 ± 0.04, n = 4, Fig. 3C) but this did not prevent their further potentiation by SNAP (popen increased further to 0.43 ± 0.05). In the presence

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Table 1 – The effects of NO on synaptic drives, spontaneous excitatory and inhibitory currents (EPSC and IPSC), and the open probability of KATP channels and L-type Ca2+ channels in the inspiratory neurons

Synaptic drives EPSC IPSC KATP channels +Hypoxia a CaL channels

Amplitude, pA Frequency, Hz Amplitude, pA Frequency, Hz Amplitude, pA Frequency, Hz popen popen

Control

NO donor (SNAP)

NOS inhibitor (NMMA)

− 35 ± 5 0.18 ± 0.03 − 35 ± 4 22 ± 3 31 ± 5 18 ± 2 0.14 ± 0.02 0.34 ± 0.03 0.28 ± 0.04

− 22 ± 5* (5) 0.12 ± 0.02* − 21 ± 3* 21 ± 2 22 ± 5* 16 ± 3 0.33 ± 0.04* (5) 0.36 ± 0.04 (4) 0.14 ± 0.03* (6)

−47 ± 6* (5) 0.28 ± 0.04* −44 ± 4* 19 ± 4 47 ± 6* 19 ± 4 0.08 ± 0.02* (5) 0.22 ± 0.02* (4) 0.38 ± 0.06* (6)

The amplitudes and the frequencies of synaptic currents were obtained as described in Experimental procedures. The data were measured at − 40 mV and are presented as mean values ± S.E.M. All values (except the frequency of spontaneous synaptic currents which showed no difference within the error of the mean) are significantly different from control values at p < 0.05 as indicated by asterisks. The number of experiments is indicated in parenthesis (synaptic drives, EPSC and IPSC were measured in the same experiments). a The activity of KATP channels measured during hypoxia.

of dithiotreitol, the hypoxic activation of KATP channels was smaller (0.24 ± 0.03 vs. 0.32 ± 0.04, n = 3), which indicated that ROS production during hypoxia (see below) can stimulate the channels. We have found any evidence for direct activation of KATP channels by NO as its application to excised patches did not change their activity (n = 6). In contrast, a relatively slow time

course of KATP channel stimulation in cell-attached patches is consistent with involvement of a multi-step cytoplasmic pathway downstream of NO that may occur by means of cGMP-dependent protein kinase. In line with this suggestion, 8-Br-cGMP enhanced the channel activity and subsequent addition of SNAP was ineffective (Fig. 3D). L-type Ca2+ channels were identified by their conductance (22 pS), voltage-dependent gating pattern and sensitivity to antagonist nifedipine and agonist Bay K 8044 (Mironov and Richter, 2000b; Mironov and Langohr, 2005). Single CaL channels were inhibited by NO donors, and NOS inhibitors potentiated the channel activity (Figs. 4A, B). t-BuHQ inhibited the channels (popen decreased from 0.32 ± 0.03 to 0.22 ± 0.04, n = 4) and the resting activity was further suppressed by SNAP (popen 0.11 ± 0.02). In the presence of 8-Br-cGMP the open probability decreased from 0.33 ± 0.04 to 0.18 ± 0.01 (n = 5), and subsequent addition of NO donors was without effect (Fig. 4C). Next, we applied fluorescent indicator dyes DAF-2 and DCF to visualize NO and ROS in the respiratory neurons. After brief (3 min) applications of hypoxia, the fluorescence of dyes increased by 53 ± 7% (n = 5, Fig. 5A) and 44 ± 6% (n = 4, Fig. 5B), respectively, which indicated enhanced production of NO and ROS. NOS inhibitors decreased the signal of DAF-2 (Fig. 5C). In the experiments, in which mitochondria were additionally stained with TMRE, we observed local elevations in the fluorescence of DAF-2 and DCF which surrounded single mitochondria (Figs. 5D and E).

3.

Fig. 2 – NO modulates the respiratory response to hypoxia. The upper and lower traces in each panel show respectively the respiratory motor output and the membrane current. The recordings were made in the control, and in the presence of NO donor (SNAP, 300 μM) and NOS inhibitor (L-NMMA, 500 μM).

Discussion

In recent years a vast evidence has been accumulated about actions of nitric oxide in the CNS which strengthened previous suggestions about the importance of NO signalling in various physiological and pathological processes (Bolanos and Almeida, 1999; Calabresi et al., 2000; Prast and Philippu, 2001; Keynes and Garthwaite, 2004; Jacintho and Kovacic, 2003; Fischmeister et al., 2005; Xu et al., 2004; Moncada and Bolanos, 2006). In brainstem, NO works as a retrograde messenger in an L -glutamate-releasing positive feedback system in NTS (Ogawa et al., 1995; Lin et al., 2000) contributing to the central

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Fig. 3 – Activation of single KATP channels by NO, ROS and cGMP. Shown are the changes in channel activity during photolysis of KRu(NO)Cl5 (0.1 mM, A), after removal of endogenous NO with scavenger PTIO (0.1 mM, B), and during applications of the oxidizing agent t-BuHQ (100 μM, C), 8-Br-cGMP (300 μM, D) and hypoxia (E). In each panel, the upper traces show continuous recordings of channel activity. The lower traces present expanded episodes at times indicated by asterisks in continuous traces. In these cell-attached recordings, the bursting activity of inspiratory neurons was suppressed by lowering extracellular K+ from 9 to 3 mM. The patch was held at 0 mV. In the presence of KRu(NO)Cl5 the slice was exposed to the light at 550 nm at the time indicated by vertical arrow. Note that SNAP increased the channel activity in the presence of t-BuHQ.

cardiovascular and respiratory control (Ling et al., 1994; Hedrick et al., 2005). We here studied the effects of NO on the respiratory rhythm which is generated within preBötzinger complex in lower brainstem (Richter and Spyer, 2001; Feldman et al., 2003) and its modulation during hypoxia. NO donors slowed down the rhythm and enhanced respiratory depression during hypoxia whereas NOS inhibitors induced opposite effects (Figs. 1 and 2). Our data extend the findings obtained in mutant mice deficient in NOS, where hypoxic responses are selectively augmented (Kline et al., 1998), indicating that endogenous NO can be important physiological modulator of respiration during hypoxia. Augmentation and depression of hypoxic respiratory response are respectively determined by CaL channels and KATP channels (Mironov and Richter, 2000a,b; Mironov and Langohr, 2005; Mironov et al., 1998, 1999). Both NO and KATP channels are also implicated in neuroprotection (Bolanos and Almeida, 1999; Keynes and Garthwaite, 2004; Sachs et al., 2004; Moncada and Bolanos, 2006). NO donors potentiated the activity of KATP channels and NOS inhibitors and NO scavengers exerted opposite effects. These actions were likely caused by inhibition of constitutive production and removal of

endogenous NO in the respiratory kernel, respectively. NOS inhibitors and NO scavengers enhanced respiration-related activities and synaptic currents, increased the activity of CaL channels and decreased the activity of KATP channels. The data add novel, functionally important targets of NO to the channels examined previously in other tissues (Nilius and Droogmans, 2001; Weiger et al., 2002; Fischmeister et al., 2005). Particularly interesting may be the effects of hemoglobin which actions as NO scavenger (Herold, 2003) can explain the differences observed in the functioning of respiratory network in vitro and in vivo (Richter and Spyer, 2001). Both pre- and postsynaptic effects of NO have been described (Calabresi et al., 2000). Presynaptic actions of NO are usually activating and postsynaptic effects are inhibitory. For example, NO suppresses GABA-receptor activated currents in cerebellum (Cupello and Robello, 2000) and increases the frequency of miniature GABAergic currents in hypothalamus (Li et al., 2004). In respiratory neurones, NO decreased the amplitude of spontaneous excitatory and inhibitory synaptic currents and did not change their frequency which indicates a postsynaptic target of NO. The time-course of synaptic currents was prolonged. KATP channels are a good candidate

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Fig. 4 – NO and cGMP suppress the activity of single L-type Ca2+ channels. Shown are the effects of NO donor SNAP (300 μM, A), NOS inhibitor L-NMMA (500 μM, B), and membrane permeable analogue 8-Br-cGMP (300 μM, C). The drugs were applied for 5 min before their effects were recorded. The channel activity was evoked by stepping the patch command potential from 0 to +60 mV which, in the cell-attached configuration, would correspond to a voltage shift from −60 to 0 mV, where activation of Ca2+ channels is maximal. Note the absence of the effects of SNAP in the presence of 8-Br-cGMP (C).

to mediate these NO actions because they are expressed postsynaptically (Matsumoto et al., 2002) and are activated by NO (Fig. 3). In this respect it is important to note that PKG postsynaptically modulates AMPA-mediated excitatory inspiratory drive currents in hypoglossal motoneurons (Feldman et al., 2005). Some actions of NO are mediated by redox reactions (Jacintho and Kovacic, 2003). We found that redox changes modified respiration-related and single channel activities indicating that CaL and KATP channels possess sulfhydryl groups which status can control the activity of the channels (Hool, 2006). In the presence of redox agents the effects of NO donors were not modified. The majority of NO effects under physiological conditions are mediated primarily by activation of guanylyl cyclase, concomitant with a cGMP increase (Bolanos and Almeida, 1999; Prast and Philippu, 2001; Keynes and Garthwaite, 2004; Moncada and Bolanos, 2006). When cGMP levels were elevated directly by 8-Br-cGMP or indirectly after inhibition of cGMP-phosphodiesterase, this suppressed respiration-related activities and NO donors showed no effect. cGMP-induced modulation of the activities of CaL and KATP channels also resembled the actions of NO donors. CaL and KATP channels are important in respiratory rhythmogenesis and its changes during hypoxia. The channels are modulated by protein kinases A and C, and Gproteins (Mironov and Richter, 2000a,b). NO- and cGMPinduced changes in channel activities observed in the present study, add novel pathway of modulation in the respiratory neurons. Ca2+-dependent NO signalling can

provide a feedback which can finely tune the activity of the respiratory network. Spontaneous activity is accompanied by Ca2+ entry which stimulates NOS and enhancement of NO production would depress neuronal activity. This can transmit changes in cell metabolism into neuronal output and vice versa. Another important NO target are mitochondrial KATP (mKATP) channels, which are activated by NO and participate in anoxic preconditioning in the heart (Lebuffe et al., 2003) and hypoxic facilitation in the respiratory network (Mironov et al., 2005). One of the important issues concerns the increase in NO concentration during hypoxia (Fig. 5). Despite this effect being observed in various cell types (Bolanos and Almeida, 1999; Keynes and Garthwaite, 2004; Henrich et al., 2004), it seemingly contradicts NO biosynthesis from L-arginine which requires the presence of oxygen. Several explanations of this apparent paradox can be proposed. For example, the lack of oxygen produces reducing atmosphere which suppresses NO destruction under anaerobic conditions that can increase NO. Elevations of intracellular Ca2+ during hypoxia (Mironov and Langohr, 2005) can stimulate the activity of NOS (Bolanos and Almeida, 1999) by overriding a deficit in oxygen. The part of NO can be produced within mitochondria (Beltran et al., 2000; Schild et al., 2003; Henrich et al., 2004) where hypoxia induces a functional switch of complex II. In addition, guanylyl cyclase can represent a “gain control” for NO signalling (Ruiz-Stewart et al., 2004): at homeostatic [ATP]i, NO activation of GC is repressed, whereas insults that reduce [ATP]i such as hypoxia derepress GC and amplify responses to NO. Which of these mechanisms dominates in the observed NO increases during hypoxia is presently unclear and needs further studies.

4.

Experimental procedures

4.1.

Preparation

The experiments were performed on medullary slices from neonatal mice (P4–9) which contained a functional respiratory network generating spontaneous oscillatory activity. The preparation was obtained following the approach developed by Smith et al. (1991) and it has been described previously in full (Mironov et al., 1998). All animals were housed and cared for in accordance with the recommendations of the European Commission (No. L358, ISSN 03786978), and the protocols were approved by the Committee for Animal Research, Göttingen University. Mice (NMRI) of both sexes were anaesthetized with ether and decapitated at the C3–C4 spinal level. A single transverse 600-μm-thick slice containing the respiratory network (pre-Bötzinger complex) was cut from the brainstem, transferred to a recording chamber and continuously superfused at 28 °C with artificial cerebrospinal fluid (ACSF, composition listed below) that was saturated with carbogen (95% O2+ 5% CO2). ACSF contained (in mM): 128 NaCl, 3 KCl, 1.5 CaCl2, 1.0 MgSO4, 21 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, pH adjusted to 7.4 with NaOH. All salts were obtained from Sigma (Deisenhofen, Germany) and NO-related drugs were obtained from Tocris Cockson.

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Fig. 5 – Imaging of NO and ROS in respiratory neurons. The slices were stained with NO- and ROS-sensitive indicator dyes DAF-2 (A, C) and DCF (B), respectively. Fluorescence images are overlaid onto the corresponding grey-scaled IR-DIC images. Note increases in DAF-2 (A) and DCF fluorescence (B) after applying hypoxia for 3 min, and decreases in DAF-2 fluorescence after addition of 0.3 mM NMMA (C). In images presented in (D) and (E), the neurons were stained with a mitochondrial marker TMRE, and DAF-2 and DCF as indicated. Green-coded images of TMRE and red-coded images of DAF-2 and DCF were merged and yellow pixels indicate TMRE-dots most which were covered by the spots of DAF-2 and DCF fluorescence. All images were background-corrected and 3 × 3 median-filtered. Calibration bar in all images = 10 μm.

4.2.

Experimental design

Slices were put on a nylon mesh in the recording chamber, overlaid with a threaded home-made platinum–iridium ‘horse-shoe’ for mechanical stability and the neurons were visualized with infrared differential interference contrast (IRDIC) illumination (Edwards et al., 1989). 30 min after the slice was positioned in the chamber, one hypoglossal (XII) rootlet

was sucked into a blunt electrode for extracellular recording of respiratory motor output. Because the reduced brainstem preparation lacks external inputs responsible for a tonic drive from the reticular formation, it was mimicked by elevating K+ concentration in the bath to 9 mM (Smith et al., 1991). For most preparations, we obtained a stable rhythm which existed up to 14 h. In this study the data were obtained from the inspiratory neurones which were collected in 42 ‘rhythmic’ slices.

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The stock solutions of drugs were made in DMSO (SNAP, diazoxide) or in ACSF (L-NMMA, PTIO, t-BuHQ, oxygenated hemoglobin, N-acetyl-cysteine, sodium nitroprusside KRu (NO)Cl5, N-ethylmaleimide and dithiotreitol) and the aliquots (at >100-fold dilution) were directly added to perfusing solution. The volume of perfusion chamber was 0.5 ml and it was renewed at a flow rate of 40 ml/min. Solutions were exchanged by replacing a distal reservoir by another one, which contained O2/N2-saturated solutions with or without corresponding drugs. New solutions arrived the slice within 10 s. The duration of the hypoxic insult was kept to minimum (3 min) so that the tissue recovered within the first minutes of reoxygenation. The responses to hypoxia were readily reproducible and they could be performed 20–30 times and after each trial the activity was completely restored. The oxygen pressure in the chamber and in the tissue was measured using oxygen-sensitive electrode (tip diameter 20 μm) and an amplifier (Diamond Electro-Tech Inc., Ann Arbor, MI, USA) as described previously (Brockhaus et al., 1993; Mironov et al., 1998). Before the measurements, the O2sensitive electrode was calibrated in two beakers with ACSF solutions saturated respectively with carbogen or a gas mixture contained 95% N2 and 5% CO2 and 1 mM Na2S2O3 to determine a zero oxygen level. During perfusion with carbogen we measured PO2 = 195 ± 25 mm Hg in the chamber which decreased to 29 ± 6 mm Hg during hypoxia. Deviation of measured levels from preset values (95% and 0% O2) were likely caused by the loss of gases during transportation of solutions and their evaporation in the open chamber. Thus, our experimental conditions corresponded to neither hyperoxia nor anoxia. In tissue measurements, the oxygen-sensitive electrode was gently driven into the slice and positioned at about 50 μm below the slice surface, within a layer, where the most inspiratory neurones were recorded. In tissue, the mean PO2 was stable and changed during hypoxia from 120 ± 25 to 21 ± 5 mm Hg (n = 7). The maximal rate of NO release from SNAP in a test tube is k = 3.10− 3 s− 1 and corresponds to activation of guanylyl cyclase (GC) with EC50 of about 2 μM (Artz et al., 2001). In tissue, NO is destructed shortly after production. Assuming the decay rate constant K = 10− 1 s− 1 (this corresponds to NO half-life time in tissue of about 7 s), the apparent EC50 for GC should be bigger by a factor K/k = 33 which gives 66 μM. Maximal GC activation would occur at about 300 μM SNAP and we used this concentration in all experiments.

4.3.

Fluorescence measurements

The slice was viewed under a 10× objective (Achroplan, N. A. 0.30) and the cells were viewed under a 63× objective (Achroplan, N. A. 0.95). The optical recording system included a Zeiss Axioscope equipped with a monochromator light source (Till Photonics, Planegg, Germany). Images from a cooled CCD camera (MicroMax, Princeton Instruments, USA) were digitized (782 × 512 pixels at 12 bit resolution) and collected with MetaMorph software (Princeton Instruments) which was also used for analysis. Changes in cytoplasmic NO and ROS (Henrich et al., 2004) were measured by using fluorescent dyes DAF-2 and carboxy-

H2CFDA (DCF), respectively, both purchased from Molecular Probes (Leiden, The Netherlands). Before each experiment, fresh solutions were made in dehydrated DMSO and the slices were incubated at 28 °C in darkness with 10 μM DAF-2/AM and DCF for 15 min and then washed with fresh ACSF for 30 min before the experiments began. The cells were illuminated at 470 nm and the emission was collected at 525 ± 10 nm. The control experiments showed that NO donors had no effect on the signal of DCF and oxidizing agents did not influence the fluorescence of DAF-2. To image mitochondria, we used a potential-sensitive dye TMRE (100 nM in ACSF) and slices were equilibrated with the dye for 30 min. After each imaging experiment, the cells in the image field were classified according to their activity in relation to the respiratory motor output. This was assessed by measuring extracellular electrical activity using a largebore pipette (inner diameter of 3 to 4 μm) which was positioned in the vicinity of each neuron in the image field. We examined only inspiratory neurons which showed the activity in phase with the motor output.

4.4.

Electrophysiology

Patch electrodes from borosilicate glass (Clark Instruments, Pangbourne, UK) had tip openings of 1.5–2 μm and resistances of 1.5–2.5 MΩ. Intracellular signals were recorded with a patchclamp amplifier EPC-7 (ESF, Friedland, Germany). Membrane currents were filtered at 3 kHz (−3 dB), digitized at 5 kHz, and stored for off-line analysis. The single-channel and whole-cell currents were measured using the amplifier EPC-7 (ESF, Friedland, Germany) as described previously (Mironov and Richter, 2000a,b; Mironov et al., 1998, 1999). Recordings of spontaneous synaptic currents in a whole-cell mode were made at the holding potential of −40 mV. In these recordings and the measurements of single KATP currents, the pipette solution contained (in mM): 125 K+-gluconate, 10 NaCl, 2 MgCl2, 10 HEPES, 0.5 Na2ATP, pH adjusted to 7.4 with KOH. The pipette solution used for single-channel recordings of CaL channels contained 110 mM BaCl2 and 10 mM HEPES, pH adjusted to 7.4 with NaOH. Inspiratory neurones were identified as those which had on-going activity correlated with the hypoglossal rhythm. Channel activity was recorded after suppression of inspiratory bursts by lowering extracellular K+ from 9 to 3 mM. The resting potentials of inspiratory neurones ranged from −55 to −65 mV (mean −60 ± 3 mV, n = 7). Because CaL channels are voltagedependent, their activity was monitored by stepping the patch command potential to the positive voltages. In terms of membrane potential, the voltage steps from 0 to +60 mV in the cell-attached configuration would correspond to the voltage shifts from − 60 to 0 mV, where activation of CaL channels reaches maximum (Mironov and Richter, 2000b). The open probability Po was obtained by dividing the mean current by the unitary current.

4.5.

Analysis of synaptic currents

Spontaneous synaptic currents are random events. We estimated their amplitude and frequency by using the extension of Campbell's theorem (Fesce, 1990) which states

BR A IN RE S E A RCH 1 1 30 ( 20 0 7 ) 7 3 –8 2

that the nth moment of randomly occurring unitary events, mn, can be expressed as Z mn ¼ f

IðtÞn dt

ð1Þ

where f is an average frequency of occurrence of the events. Approximating synaptic currents by alpha function (Ulrich and Lüscher, 1993) IðtÞ ¼ Qb2 t expðbtÞ

ð2Þ

where I(t) and Q are the current and the charge crossing a postsynaptic membrane, and β is the time constant. The two first moments, the mean and the variance, are m1 ¼ fQ m2 ¼ f bQ 2 =4

ð3Þ

The mean amplitude and frequency of synaptic currents were determined as Q ¼ 4m2 =bm1 f ¼ bm21 =4m2

ð4Þ

Each test performed in this study was repeated for at least three different slice preparations and means ± S.E.M. were compared by using Student's t test, with p < 0.05 being the criterion for statistical significance. The analysis of variance (ANOVA) was used for multiple comparisons.

Acknowledgments The authors thank N. Hartelt for excellent technical assistance.

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