brain research 1543 (2014) 9–16

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Endogenous nitric oxide but not exogenous no-donor S-nitroprussiate facilitates NMDA excitation in spontaneous rhythmic neonatal rat brainstem slice Olivier Pierrefichen, Mickael Naassila INSERM ERi 24 GRAP, Groupe de Recherche sur l’Alcool et les Pharmacodépendances, UFR de Pharmacie, 1 rue des Louvels, 80036 Amiens, France

art i cle i nfo

ab st rac t

Article history:

Nitric oxide (NO) is an excitatory agent within the isolated respiratory network of immature

Accepted 22 October 2013

rats (Pierrefiche et al., 2002) and modulates bursting discharge of rhythmic respiratory

Available online 29 October 2013

neurone in juvenile rats (Pierrefiche et al., 2007). However, whether NO is acting directly via a

Keywords:

specific cellular mechanism or by increasing NMDA receptor activity is unknown. Our

Nitric oxide

present aim was to study NO modulation of NMDA-induced excitation within the isolated

NMDA

neonatal respiratory network. The NO-scavenger, haemoglobin, and the NOS inhibitor L-NO-

Respiratory

Arg, reduced spontaneous activity and were more effective during NMDA-induced excita-

Ferrocyanide

tion. Both diethylamine-NO (DEA-NO) and S-nitroprussiate (SNP), two NO-donors not related

SNP

chemically, increased spontaneous activity in a dose-dependent manner. However, when co-applied with NMDA only DEA-NO facilitated NMDA-induced excitation whereas SNP partially reversed or prevented NMDA-induced excitation. Similar reversion of NMDAinduced excitation were obtained with K3–(FeCN)6 (Fe III) or inactivated SNP. On the contrary, FeSO4 did not have any effect on either spontaneous activity or NMDA-induced excitation. These data suggest that activation of NMDA receptors increase endogenous NO production which participates in endogenous NMDA-induced excitation during spontaneous XII bursting activity. It also demonstrated that the type of NO-donors used during pharmacological study implicating NMDA receptors should be carefully chosen. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Endogenous glutamatergic neurotransmission participate in rhythmic bursting activity of respiratory neurones of the ventrolateral medulla both in vivo and in vitro, in neonates and adults rodents and feline animals (Bianchi et al., 1995). Stimulation of the N-methyl-D-aspartate (NMDA) type of glutamate receptors is particularly involved along with non-NMDA receptors (Funk n

et al., 1993; Pierrefiche et al., 1991, 1994). This endogenous excitation allows the transmission of the respiratory central command to final effectors for breathing (i.e. respiratory muscles). Among the effect of NMDA receptor activation, it is known that influx of calcium through this receptor promotes nitric oxide (NO) production by activation of the neuronal NO synthase isoform (Kato and Zorumski, 1993). In previous studies we showed that exogenous as well endogenous NO

Corresponding author. Fax: þ33 3 22 82 76 72. E-mail addresses: [email protected], olivier.pierrefi[email protected] (O. Pierrefiche).

0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.10.042

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facilitate bursting rhythmic activity of the hypoglossal nerve recorded from spontaneous rhythmic slice of neonatal rat, that respiratory areas contained in the slice are stained for NADPH-diaphorase activity (Pierrefiche et al., 2002) and that bulbar respiratory neuronal activity in an in situ preparation was increased in presence of exogenous NO (Pierrefiche et al., 2007). The cellular mechanism(s) of endogenous NO within the respiratory network is, however, still unknown. The increase in XII burst amplitude reported previously (Pierrefiche et al., 2002) may have come, among other type of mechanisms, from a facilitation of glutamatergic neurotransmission and/or a decrease in inhibitory neurotransmission. NO has been reported to modulate glutamatergic neurotransmission in different models, from cells culture to anaesthetized animals by either increasing or decreasing glutamatergic transmission and/or by modulating NMDA receptor openings (Shaw and Salt, 1997; Cox and Johnson, 1998). In the present study we tested the hypothesis that NO facilitates NMDA-induced excitation within the isolated respiratory network of neonatal rat studied in vitro. We previously tested two different types of NOdonors which belong to different chemical family (Pierrefiche et al., 2002, Pierrefiche et al., 2007): DEA-NO, a derivative of ethylamine from the NONOate family and, S-nitroprussiate (SNP). Although both substances deliver NO, SNP is also known to release different types of iron derivative ions once in solution which are capable of inhibiting NMDA receptors (Manzoni et al., 1992; Neijt et al., 2001). Studying the interaction between NMDA-induced excitation and these NO-donors lead to the hypothesis that endogenous and DEA-NO might facilitate NMDA-induced excitation whereas SNP should reduced excitatory effects of NMDA, due to its by-product.

concentrations tested. These results are in accordance with our previous report (Pierrefiche et al., 2002).

2.2. Involvement of endogenous NO in NMDA-induced excitation There are several possible mechanisms to explain the mode of action of NO as an excitatory agent within the respiratory network. One of these mechanisms could be through an interaction with endogenously active NMDA receptor. We thus determined whether endogenous NO interacted with NMDAinduced excitation. For this purpose, we applied haemoglobin, an NO-scavenger or L-nitro-arginine (L-NO-Arg), a competitive inhibitor of NO synthesis, during NMDA-induced excitation assuming that their respective effect should be stronger once NMDA-induced excitation are increased. When applied alone, these two substances inhibit the amplitude of spontaneous XII bursting activity by about 10% and 20%, respectively (Pierrefiche et al., 2002). NMDA applied at 10 mM, increased significantly XII burst amplitude by 51.3718.9% (Po0.01, n¼ 6) without affecting burst frequency. In 4/4 slices tested, application of haemoglobin (Hb, 3 mg/L) during NMDA effects, reduced NMDA-induced excitation on burst amplitude by 30.376.1% (Po0.05; Fig. 2A and B) and co-application of L-NO-Arg on 2/2 slices tested also decreased NMDA-induced augmentation by a similar amount (35.372.4%, Po0.05). The inhibitory effects of these two drugs on NMDA-induced excitation were similar (Fig. 2B, P40.05 between L-NO-Arg and Hb effects). This series of experiments suggested that NMDA receptors activation was responsible of an increase in NO levels which in turn participate in NMDAinduced excitation and/or spontaneous rhythmic activity.

2.3. DEA-NO applications and co-applications with NMDA

2.

Results

2.1. Effects of NO-donors on spontaneous rhythmic activity SNP and DEA-NO, two exogenous NO-donors not chemically related were tested at different concentrations (Fig. 1A and B). Both substances induced similar increase in XII burst amplitude without effects on rhythmic frequency. DEA-NO (Fig. 1) tested in control conditions (i.e., on spontaneous rhythmic activity) was applied for 8–10 min, at 50 mM (n¼ 8), 100 mM (n ¼8) and 200 mM (n ¼17). Excitatory effect of DEA-NO developed rapidly to reach a maximum after 6–8 min of application. At 50 mM, DEA-NO induced a significant increase in XII burst amplitude (21.878.5%, Po0.05, n ¼7). At 100 mM, DEANO increased significantly the amplitude by 28.576.5% (Po0.05, n ¼6). At 200 mM a greater increase of the burst amplitude was obtained (40.077.1%, Po0.01, n¼ 17; Fig. 2B). The effects of DEA-NO on frequency were variable at all concentrations tested and never reached statistical significance. SNP (Fig. 1A and B) bath applied for 10–15 min in the same range of concentration induced similar increase in spontaneous XII nerve amplitude. Amplitude was increased by 28.3712.6% at 50 mM (Po0.05, n ¼7), 34.077.9% (Po0.05, n¼ 9) at 100 mM and, 48.7715.4% (Po0.01, n ¼9) at 200 mM (Fig. 1B). SNP effects on frequency were not significant at all

In order to clarify the type of interaction between NMDA receptors and NO, we tested first the exogenous nitric oxide donor DEA-NO during NMDA-induced excitation. We first tested both substances at threshold concentrations (Fig. 3A). NMDA (1.5 mM) elicited a significant increase in XII burst amplitude (þ22.774.7%, Po0.01, n¼ 9), while frequency was not modified. DEA-NO (50 mM) applied together with NMDA (1.5 mM) increased burst amplitude by a mean of 82.3710.8% (Po0.01, n¼ 9). This effect was greater than NMDA or DEA-NO applied alone (unpaired t-test, Po0.05), suggesting a facilitation of NMDAinduced excitation by DEA-NO. Furthermore, this facilitation was also observed for higher concentration (data not shown, DEA-NO 200 mM and NMDA 5 mM). At higher concentration, however, the effect of DEA-NO was particularly visible in the increase of tonic activity induced by NMDA at 5 mM.

2.4.

SNP applications and co-applications with NMDA

We wished to check whether both type of NO-donors induced the same type of effects on NMDA-induced excitations because these substances are not chemically related and because the literature is unclear regarding NMDA/NO-donors interaction (Gbadegesin et al., 1999; Cudeiro et al., 1996; Manzoni et al., 1992; Neijt et al., 2001). Surprisingly, when SNP was applied during NMDA-induced excitation, the increase in XII burst

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SNP 200 µM + 30 min 100 % Int. XII 0 1 min

DEA-NO 50 µM

100 % Int XII 0

15 min

1 min

% increasein XII amplitude

** ** *

* *

*

50 µM

100 µM

200 µM

DEA-NO

SNP

Fig. 1 – Exogenous NO and XII burst activity. (A) Example of the excitatory effects of SNP on XII burst activity (top traces) and of DEA-NO (lower traces), two non-chemically related NO-donors. (B) Bar graph illustrating the responses for the two exogenous NO-donors on XII burst amplitude. All results are significant with (*) for Po0.05. Int. XII: integrated form of XII nerve activity.

NMDA 10µM Hb3mg/L

Hb3mg/L

100% Int. XII 0 2 min

Fig. 2 – Endogenous NO participates in NMDA-induced excitation. (A) Haemoglobin, a scavenger of endogenous NO, decreased NMDA-induced excitation. This effect was reversible at the end of Hb application and can be repeated during continuous NMDA application. (B) Bar graph summarizing the effects of Hb and L-nitro-arginine (L-NO-Arg), a competitive inhibitor of NO synthesis, during NMDA application which elicited a similar effect than Hb. Same abbreviations as in Fig. 1.

amplitude induced by NMDA was partially and rapidly reversed (Fig. 3B). At threshold concentration, NMDA 1.5 mM increased burst amplitude by 19.171.2%, (Po0.05, n ¼5).

However, co-applications of NMDA (1.5 mM) with SNP 50 mM abolished the excitatory effect of NMDA ( 6.575.5%, P40.05, n¼ 5). We next checked whether this inhibitory effect of SNP,

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Fig. 3 – Interaction between NO donors and NMDA. (A) Interaction between 50 lM DEA-NO and, 1.5 lM NMDA revealing a potentiating effect of NO on NMDA-induced excitation. (B) Interaction between SNP and NMDA 5 lM showing the inhibitory effect of SNP on NMDA-induced excitation. The bar graph illustrates the population tested. Interestingly, inactivated SNP was more efficient in inhibiting NMDA excitations than SNP. (C) Inhibitory effect of inactivated form of SNP at 20 lM. Same abbreviations as in Fig. 1.

in contradiction with its excitatory effect when applied alone, was dependent of the level of excitation by increasing drug concentrations. The excitation induced by high concentration of 5 mM NMDA (32.773.5%, Po0.01, n¼ 9) was also significantly reduced by SNP (200 mM) by a mean of  26.674.0% (n ¼10, Po0.05; Fig. 3B). Furthermore, when applied simultaneously SNP prevented the NMDA excitatory effect (data not shown). One way to confirm that such inhibition was due to NO delivered by SNP is to test its inactive form (see Section 5) which is devoid of NO delivery. Interestingly, inactivated SNP (200 mM) was not excitatory but indeed reduced spontaneous activity of XII nerve rootlet by a mean of 31.977.3% (Po0.05, n¼4, Fig. 3C). When co-applied with NMDA (5 mM) or applied during NMDA (Fig. 3B), NMDA effect was inhibited in a stronger way than that with active form of SNP ( 42.676.0%, Po0.05, n¼ 5 and Po0.05 between SNP and inactivated SNP, Fig. 5B). This result suggested that the inhibitory effect of SNP on NMDA-induced excitation was due to SNP photolysis by-products rather than NO.

2.5. Involvement of SNP by-products in the inhibition of NMDA-induced excitation The differential effect of the two classical NO-donors on NMDAinduced excitation suggested that by-product of SNP was involved in the inhibitory effect of SNP. To determine the potential role of the photolysis by-products of SNP (i.e., ferrocyanide/ferricyanide ions), we applied in control period and during NMDA effects the potassium salt of ferricyanide ions: K3–

(FeCN)6. We therefore tested the possibility that NMDA response was indeed depressed by this photolysis by-product of SNP. In control conditions, application of 200 mM K3–(FeCN)6 significantly decreased XII burst amplitude ( 15.573.4%, Po0.01, n¼7; Figs. 4A and 5A) without effects on burst frequency. This inhibitory effect of K3–(FeCN)6 was abolished in 2/2 cases tested by prior application of AP-5 (10 mM), a competitive antagonist of NMDA receptors. In presence of AP-5, K3–(FeCN)6 inhibitory effect on XII bursting activity reached 6.172.1% (P40.05). Application of K3-(FeCN)6 at 200 mM in presence of NMDA (5 mM), significantly reversed NMDA-induced excitation by –19.177.3% (Po0.05, n¼ 7) (Figs. 4A and 5B). Moreover, when both drugs were co-applied, NMDA-induced excitation was totally prevented (n¼ 6). Recovery of the NMDA response from K3–(FeCN)6 effects took 20–30 min washout. This inhibitory effect of K3–(FeCN)6 was, however, not observed with FeSO4 (200 mM, Fig. 4D) co-applied together with NMDA ( 9.4718.5%, P40.05, n¼ 4, Fig. 5B) revealing that ferricyanide ions are responsible for NMDA inhibition. Furthermore, 200 mM FeSO4 did not modify spontaneous activity of XII bursting on either amplitude or frequency (1.473.6% for amplitude and 0.0571.9% for frequency, both P40.05, n¼ 6, Fig. 5A).

3.

Discussion

The present results suggest that endogenous nitric oxide (NO) is produced once NMDA receptors are activated and participates

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NS

** *

% inhibition of NMDA response

% inhibition of XII amplitude

Fig. 4 – Effects of ferricyanide ions on NMDA-induced excitation. (A) 200 lM K3–Fe(CN)6 applied alone, inhibited the XII bursting activity. (B) During 5 lM NMDA application, 200 lM K3–Fe(CN)6 reversed NMDA-induced excitation in a repeatable manner along NMDA application. (C) When co-applied, NMDA-induced excitation was totally prevented. (D) The graph for the whole applications performed illustrates the effect of K3–Fe(CN)6 and the lack of effect of FeSO4 on NMDAinduced excitations.

*

**

NS

* *

Fig. 5 – Summary of drug effects. (A) All tested drugs except FeSO4 decreased spontaneous XII burst amplitude. (B) NMDAinduced excitations were reduced in presence of all drugs except FeSO4. Interestingly, inactivated form of SNP reduced significantly more the NMDA excitation than active form of SNP. nPo0.05; nnPo0.01.

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in the modulation of NMDA-induced excitation and thus spontaneous glutamatergic excitation during spontaneous bursting activity. Our results further demonstrate that exogenous NO has different effects on NMDA-induced excitation according to the chemical family the NO-donor belong to. Indeed, we showed that photolysis by-products of SNP are responsible for the inhibition of NMDA receptors activation rather than NO per se in a spontaneous rhythmic neuronal network. Endogenous activation of NMDA receptors is an essential component of respiratory rhythmic activity in neonates and adult mammals studied both in vivo and in vitro (Funk et al., 1993; Pierrefiche et al., 1991, 1994). Activation of this glutamatergic ionotropic receptor has been shown in other parts of the brain to induce a production of NO through the activation of the neuronal isoform of NOS which is calcium-dependent (Kato and Zorumski, 1993). Once produced, NO may have several biological effects because of its numerous cellular targets including ionic channels, enzyme, and neurotransmitter release processes. There is also an endogenous production of NO modulating XII rhythmic activity in neonatal rat respiratory network (Pierrefiche et al., 2002) or other cranial nerves related to breathing in lower vertebrate species (Hedrick and Morales, 1999) studied with in vitro preparations. Furthermore, in the respiratory network, NO is facilitating the active phase of some respiratory bulbar neurons (Pierrefiche et al., 2007). Our results showed that within isolated respiratory network from neonatal rats, NMDA is responsible for a production of NO since NMDA excitatory effects were reduced in presence of an NO-synthase blocker. In addition, NO once produced facilitates glutamatergic transmission because haemoglobin, an NO trap agent, also reduced NMDA-induced response on spontaneous XII rhythmic activity. This latter suggestion is further supported by the fact that both haemoglobin and L-NO-Arg were more effective during NMDAinduced excitation than during spontaneous rhythmic activity, indicating an increase in NO production during NMDA receptor activation. Both substances inhibited spontaneous rhythmic activity by about 10% and 20% (Pierrefiche et al., 2002) and they both reversed NMDA-induced excitation by about 35%. These results suggest that NO reinforced NMDA receptors activation within the neonatal respiratory network in vitro. Several reports described an inhibition or a reinforcement of NMDA-receptors or NMDA-induced responses by the nitrergic route. In vivo blockade of NOS accelerated the establishment of amygdala kindling, an NMDA-dependent phenomenon (Rondouin et al., 1992); L-arginine, the substrate of NOS, reduced NMDA receptor effect on primary culture of striatal neurones (Manzoni and Bockaert, 1993) while L-NAME, a non-specific NOS inhibitor, reduced NMDA-induced burst firing in dopaminergic neurones in rat midbrain slices (Cox and Jonhson, 1998). Dual excitatory and inhibitory effect of SNP has also been reported in the induction of synaptic longterm potentiation in mouse hippocampal slice (Li and Wieraszko, 1994) through the modulation of the NMDA component of the population spike. Interestingly, the present results also showed a blockade of NMDA-induced excitation within the respiratory network of neonatal rat by SNP, a classically used NO-donor (Saransaari and Oja, 2003; Ryu et al., 2001; Chen et al., 2001, Lin et al., 1999). The herein

reported effects of SNP on NMDA-induced excitation are contrary to what expected from an NO-donor (Gbadegesin et al., 1999) and are contrary to the potentiating effect of SNP on feline lateral geniculate nucleus (Cudeiro et al., 1996). However, the present blocking effect of SNP is unlikely to be due to NO release since it was mimicked by ferricyanide ions, structurally related to SNP but devoid of NO release properties, and furthermore by inactivated-SNP which is unable to release NO (Neijt et al., 2001). In addition, FeSO4, which do not contain cyanide group, was unable to affect either spontaneous or NMDA-induced activity at the level of XII bursting amplitude or frequency. The effect of ferricyanide ions on NMDA-induced excitation is in agreement with previous study from Manzoni et al. (1992) although they studied ferrocyanide ions (K4–FeCN). Oh and McCaslin (1995) give precision on the effects of the different iron ions derived from cyanide compounds. They found that ferricyanide ions did not blocked NMDA-induced cGMP accumulation and intracellular calcium elevation on granular cell culture, on the contrary to ferrocyanide ions. However, Ogita et al. (1998) showed that potassium ferrocyanide and ferricyanide both inhibits MK-801 binding on the NMDA recognition domain like SNP do. This is supported by the study of Shuto et al. (1997) who suggest that ferrous ions interfere with the opening processes of NMDA channels while ferrique ions inhibit NMDA receptor function in a different and still unknown manner. Indeed, Neijt et al. (2001) reported that photodegradation products of SNP represent a new family of selective and competitive antagonists of NMDA receptors which interact with the receptor through their highly negative charge. Our results are in accordance with Neijt et al. (2001) since coapplied SNP and NMDA result in a blockade of NMDAinduced excitation. In contrast, the lack of inhibitory effect of FeSO4 in the present study and others (Manzoni et al., 1992) could possibly be attributed to its positive charge once in solution (Neijt et al., 2001). Also, effects of SNP on expressed NMDA receptors into oocytes do not show any interaction with the NMDA receptor redox site (Omerovic et al., 1994). In summary, the inhibitory effects of SNP on NMDA-induced excitation has been reproduced by co-applications of NMDA with either K3–(FeCN)6 or inactivated-SNP, but not with FeSO4, revealing that inhibitory effects of SNP on activated NMDA receptors were mainly due to ferricyanide ions rather than cyanide or NO. At first, the present results may seem surprising since we reported that these two different NO-donors increased similarly the amplitude of XII bursting activity in neonatal rat rhythmic slices reproducing then our previous findings (Pierrefiche et al., 2002). In the present study SNP was quite potent in reducing NMDA-induced excitation whereas in control period (i.e. without NMDA applications), it increased XII burst amplitude. Therefore, SNP effects seem to depend on the level of NMDA-induced excitability within the respiratory network. At low level of excitation (i.e. endogenous glutamatergic neurotransmission), SNP is able to increase XII burst amplitude while at higher level of excitation (exogenous NMDA applications), it turns out that cyanide ferrous compounds from SNP take an important role. This discrepancy in SNP effects was probably by a direct action on the receptor channels (Neijt et al., 2001). Increased excitability

brain research 1543 (2014) 9–16

with NMDA would also probably increase its own blockade by SNP since exposure to glutamate exacerbate the inhibitory effect of SNP on glutamate binding in rat brain synaptic membranes (Ogita et al., 1998), suggesting a biphasic effect of SNP. We may therefore hypothesise that SNP effects on spontaneous activity of respiratory-related nerves have a different mechanism(s) that was observed during increased excitability. We believe that the most likely possibility is that the effects of NO-donors on NMDA receptors differ according to the chemical family to which the NO-donor belongs. Manzoni et al. (1992) described an inhibitory effect of SNP on NMDA-induced excitation due to ferrocyanide ions, an effect that was reproduced in the present study. Shaw and Salt (1997) described that SNP reduced NMDA-induced neuronal discharge in the thalamus of the rat, an effect not obtained with other NO-donors (S-nitrosoglutathione or 3-morpholinosydnonimine) which caused a potentiation, although others showed an inhibition of NMDA-induced responses with morpholinosydnonimine (Manzoni et al., 1992). Finally, Gbadegesin et al. (1999) demonstrated that diethylamineNO (DEA-NO), from the NONOate family increased the open probability of the NMDA receptor channel. It is thus conceivable that in our experimental model, SNP-induced excitation on spontaneous XII rhythmic activity may come at least partly from an interaction of NO with NMDA receptors, reinforcing glutamatergic neurotransmission. However, we did not record cellular rhythmic discharge, the release of neurotransmitters, or accumulation of cGMP and thus, we cannot localise whether the facilitating effect of NO is at pre- or postsynaptic site or direct/ indirect on the receptor itself. Another possible effect of SNP could also be via an inhibition of D-serine release from astrocytes (Mustafa et al., 2007). These present results further support the evidence that NO is responsible for an excitation of respiratory neurones within neonatal rat rhythmic slice and illustrates that SNPinduced excitation of basal XII bursting activity, whatever would be the cellular mechanism, completely masked the inhibitory effects of ferricyanide ions released within the recording chamber during SNP applications. Finally, the present results further document that the use of SNP as a NO-donor in studies concerning the central nervous system, especially where NMDA receptors have a crucial role (Busnardo et al., 2013; Martins-Pinge et al., 2012; Sun et al., 2012, Jin et al., 2012; Tozer et al., 2012; Gourgiotis et al., 2012), or when SNP effects are opposite to those of other NO-donors (Saransaari and Oja, 2008) is not an appropriate substance. It would be more prudent to use one NO-donor which does not content FeCN chemical groups or to check carefully whether by-products of SNP play a role in the reported results.

4.

Conclusion

Our results suggest that endogenous nitric oxide is produced once NMDA receptors are activated and increases respiratorylike activity in neonatal rat rhythmic slice via potentiation of NMDA receptors activity. The present results also demonstrate the necessity to use NO-donor devoid of by-products, notably containing FeCN chemical groups, in studies concerning the NO–NMDA interaction.

5.

15

Experimental procedures

The procedures described are in accordance with the guidelines for care and use of laboratory animals adopted by the European Community, law 86/609/EEC. Rhythmic brainstem transverse slices were obtained from neonatal rats (aged P3–P6) anaesthetized with ether and then decapitated. The brainstem was removed in artificial cerebrospinal fluid (aCSF) continuously oxygenated with carbogen (5% CO2; 95% O2; pH 7.4) and then cut serially in the caudal direction with a vibroslicer. Rhythmic slices were obtained 300–500 mm caudal to the inferior anterior cerebellar arteries (700–750 mm thickness). The slice was put in a recording chamber continuously superfused with oxygenated aCSF (27.5–28 1C) of the following composition (in mM): NaCl 126, KCl 5, NaHCO3 21, NaH2PO4 0.5, MgSO4 1, CaCl2 1.5, glucose 30, and at a rate of 15 ml/mn. Extracellular XII nerve rootlet and/or pre-Bötzinger mass activity were recorded with the use of suction electrodes. Raw activities were amplified (50,000 times) and integrated (τ¼100 ms), and all signals were continuously recorded through a CED 1401 interface (Spike3 acquisition program, Cambridge, U.K.) connected to a personal computer.

5.1.

Drug solutions

All drugs were obtained from Sigma inc. (France). Haemoglobin (Hb) and L-nitro-arginine (L-NO-Arg) were prepared in aCSF. Stock solutions of NMDA were prepared in saline. DEA-NO was first dissolved in 0.1 M NaOH, and final concentration was obtained by further dilution in aCSF. Potassium ferricyanide (K3–(FeCN)6), FeSO4 and S-nitroprussiate (SNP) were prepared as stock solutions in aCSF or distilled water. SNP and potassium ferricyanide were protected from direct light exposure and applied in the dark. In some experiments we applied inactivated SNP obtained after several hours of exposure to daylight (Neijt et al., 2001). Drugs were prepared at final concentrations in a reservoir containing oxygenated and equilibrated aCSF maintained at the same temperature than the main reservoir and applied through a tab connected to the main flow line. Washout was performed by passing drug-free aCSF throughout the recording chamber.

5.2.

Data analysis

Off-line analysis consisted in measuring the amplitude and frequency of 10 to 20 consecutive cycles on their integrated form, before, at the end of drug application and after recovery. Values obtained were then averaged and Student t-test performed between relevant groups. Statistical significance was considered for Po0.05. Values are presented as group mean7S.E.M.

r e f e r e nc e s

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Endogenous nitric oxide but not exogenous no-donor S-nitroprussiate facilitates NMDA excitation in spontaneous rhythmic neonatal rat brainstem slice.

Nitric oxide (NO) is an excitatory agent within the isolated respiratory network of immature rats (Pierrefiche et al., 2002) and modulates bursting di...
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