BJA Advance Access originally published online on January 23, 2006
British Journal of Anaesthesia 2006 96(3):361-366; doi:10.1093/bja/ael010
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Inhibition of neuronal nitric oxide synthase reduces isoflurane MAC and motor activity even in nNOS knockout mice
Academic Unit of Anaesthesia and Intensive Care, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
* Corresponding author. E-mail: t.engelhardt{at}abdn.ac.uk
Accepted for publication December 1, 2005.
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Abstract |
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Background. The glutamate–nitric oxide–cyclic GMP pathway has been identified as a potential target for volatile anaesthetic agents as acute inhibition of nitric oxide synthase (NOS) reduces the minimum alveolar concentration (MAC) in most animal studies. However, mice deficient in the type I NOS isoform (nNOS) are reported to have a similar MAC for isoflurane and are not affected by non-isoform specific inhibitors.
Methods. We determined whether the nNOS specific inhibitor, 7-nitroindazole (7-NI), had an effect on isoflurane MAC and righting reflex (RRF) and investigated spontaneous motor activity in an open-field study in wild-type (WT) and knockout (KO) mice.
Results. 7-NI reduced isoflurane MAC and RRF in both WT and KO animals (all P<0.04). 7-NI profoundly reduced spontaneous motor activity in both the WT and KO animals in the open-field study as indicated by a reduction in the number of line crossings and rearings in both WT and KO mice (both P<0.001).
Conclusion. We conclude that isoform specific inhibition of nNOS reduces MAC and spontaneous motor activity even in nNOS KO animals. Our results indicate that the NMDA receptor–nitric oxide–cyclic GMP pathway remains a credible target in modulating the effects of isoflurane.
Keywords: anaesthetics volatile, isoflurane; inhibitor, 7-NI; isoform, nNOS; mice, open-field
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The N-methyl-D-aspartate (NMDA)–nitric oxide (NO)–cyclic GMP pathway has a potential role in modulating general anaesthesia as inhibition of nitric oxide synthase (NOS) by means of selective and non-selective isoform inhibitors leads to a reduction of minimum alveolar concentration (MAC) for most volatile anaesthetic agents.1–7 However, a single report using gene deleted mice, deficient in the neuronal isoform (type I) of NOS (nNOS) showed no altered susceptibility to isoflurane in the presence or absence of a non-isoform specific inhibitor (L-NAME)4 and this diminished interest in this potential pathway.
It has since been demonstrated that only the major nNOS
isoform is absent in these knockout (KO) mice and alternative nNOS mRNA splice variants are up-regulated accounting for an observed residual 5% brain NOS activity.8–10
7-Nitroindazole (7-NI) is a nNOS specific inhibitor and reduces MAC for the volatile agents sevoflurane and isoflurane in mice and rats.3 5 7 7-NI also reduces spontaneous motor activity in mice and may have sedative and anxiolytic properties necessitating the investigation of the effects of 7-NI in the non-anaesthetized animal.11 12 We hypothesized that the nNOS specific inhibitor 7-NI would reduce isoflurane MAC and spontaneous motor activity in wild-type (WT) mice but would have a limited effect in nNOS KO mice.
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Materials and methods |
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The studies were approved by the United Kingdom Home Office under the Procedures of the Animals (Scientific Procedures) Act 1986.
Animals
A total of 48 animals (24 WT and 24 KO) were used in the studies. Animals were bred from imported breeding pairs (B6;129S-Nos1tm1Plh, stock number 002633 for nNOS KO mice and B6129SF2/J, stock number 101045 for WT controls; Jackson Laboratories, Bar Harbor, USA). They were housed in a 12 h light–dark cycle, with free access to feed and water. Animals aged 10–14 weeks weighing 21–31 g were randomly selected from male and female cages.
The presence of the neomycin resistance gene in place of exon 2 of the nNOS gene in KO mice and the presence of the exon 2 of nNOS gene in the WT animals was confirmed by polymerase chain reaction (PCR) from DNA obtained from tail clippings. The last 2–3 mm of mouse tails were incubated in a buffer [KCl 50 mM, Tris–HCl 10 mM (pH 8.3 at 22°C), 2.5 mM MgCl2, gelatin 0.01% w/v, Nonidet P40 0.45% v/v, Tween 20 0.45% v/v and proteinase K 2% w/v] for 3 h at 55°C and for 10 min to 95°C for inactivation of proteinase K.
The oligonucleotide primers (MWG Biotech, Ebersberg, Germany) for the nNOS coding region used were:
- B1 primer: 5' CCT TTG AGA GTA AGG AAG GGG GCG GG 3' (26mer)
- B2 primer: 5' GGG CCG ATC ATT GAC GGC GAG AAT GAT G 3' (28mer)
- B2 primer: 5' GGG CCG ATC ATT GAC GGC GAG AAT GAT G 3' (28mer)
They amplify a region between nucleotides 156 and 561 of the coding region of the nNOS gene and give a 404 bp PCR product in WT mice. The primers for the neomycin resistance gene used were:
- CFA: 5' ATG AAC TGC AGG ACG AGG CAG CG 3' (23mer)
- CFB: 5' GGC GAT AGA AGG CGA TGC GCT G 3' (22mer)
- CFB: 5' GGC GAT AGA AGG CGA TGC GCT G 3' (22mer)
These primers amplify a 603 bp product in the KO mice.
The PCR reaction [1 µl DNA template, PCR buffer (MgCl2 1.5 mM, Tris–HCl 10 mM and KCl 50 mM final), B1, B2, CFA, CFB primers 10 µM each, dNTP 12.5 mM, Taq 1 U and double distilled water to a final volume of 50 µl] was incubated as follows: (i) 94°C 3 min, (ii) (94°C 0.5 min, 61°C 0.5 min, 72°C 1 min) cycle 30 times, (iii) 72°C 3 min. PCR products were analysed on a 1.5% agarose gel and stained with ethidium bromide for visualization under UV light (Fig. 1).
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7-Nitroindazole and L-arginine pre-treatment
7-NI was suspended in arachis oil, and sonicated at 4°C before intraperitoneal (i.p.) injection (120 mg kg–1). All animals were then kept warm in a heated incubation chamber to a rectal temperature of 37°C for 1 h. All animals had maintained their righting reflex (RRF) after the incubation period but appeared to have a decreased spontaneous motor activity and broad based gait. The decreased spontaneous motor activity was subsequently quantified in an open-field study. Untreated animals served as controls.
The effects of 7-NI can be at least partially reversed by co-administration of the amino acid L-arginine, which also applies to most non-specific NOS inhibitors (for example L-NMMA and L-NAME). We used 600 mg kg–1 i.p. 1 h after 7-NI injection as previously described.5 Correct i.p. injection was confirmed post-mortem.
Determination of isoflurane MAC and RRF
Determination of isoflurane MAC and RRF values was carried out as previously described.5 6 Briefly, WT and KO mice (n=6 per group) were placed into a custom built Plexiglas chamber and anaesthesia was induced with 2% isoflurane in oxygen at 2 litre min–1 through a regularly calibrated TEC3 isoflurane vaporizer. Isoflurane concentration was continuously monitored at the Plexiglas chamber gas outlet using an agent analyser, Capnomac UltimaR (Datex-Ohmeda, Helsinki, Finland). Rectal temperature was kept at 36–38°C throughout the experiment.
After an initial equilibration period of 30 min, MAC was determined by tail clamping of the middle third of the tail to the first ratchet of a 15 cm haemostatic forceps for up to 1 min. Gross motor movement was judged to be a positive response, and no movements or grimacing and swallowing were judged a negative response. If a negative response was observed, isoflurane concentration was reduced by 0.1% for 10 min. If a positive response was seen isoflurane concentration was increased by 0.1% for 10 min. The midpoint isoflurane concentration for negative and positive responses was taken as MAC. The initial equilibration period for the next animal was the previous MAC+isoflurane 0.2%.
After determining MAC in each animal, the isoflurane concentration was halved for 10 min and the animal turned on its back to test the RRF defined as a return onto all four paws within 60 s. The isoflurane concentration was reduced by 0.1% for 10 min if the animal failed to right itself and the RRF subsequently re-tested. After completion of experiments, the animals were killed by rapid cervical dislocation. The Plexiglas chamber was cleaned with 5% ethanol between experiments.
Open-field studies
An open-field system was used to investigate 7-NI induced motor activity, this consisted of a box appropriate to the animal size with its floor space divided into equal areas. The movement of the animal from one area to another was monitored over a fixed time period. This method is sensitive enough to observe changes in spontaneous activity, can be automated and continuously applied.11 13
Twelve new WT and KO mice (n=6 per group) were placed in the same starting square in an open-field box (30 cmx30 cmx40 cm, grey metal marked into four squares 15 cmx15 cm) with a light source (60 W at 100 cm distance) for 4 min the day before experimentation to allow familiarization with the environment.11 The following day all mice were again exposed to the open-field box for 4 min and the number of line crossings, rearings and excretions were noted. A line was judged to have been crossed once all four paws were inside the same quadrant. Rearing was defined as the animal standing on the hind paws with or without touching the sidewalls of the box for at least 1 s. All four paws had to touch the floor of the box again before a new rearing could be achieved. Stool pellets and number of urinations were counted for excretions. All animals were then injected i.p. with 7-NI (120 mg kg–1) 1 h before re-exposure to the box. Line crossing and rearing were determined immediately before and 1, 2, 3, 4 and 6 h after 7-NI injection. All animals were kept warm in a heated incubation chamber between exposures with access to water ad libitum. The open-field box was cleaned with 5% ethanol between experiments.
Statistical analysis
Data were not normally distributed and are presented as median and range. Statistical analysis of MAC and RRF experiments was performed using Kruskal–Wallis 1-way ANOVA and Mann–Whitney U-test as appropriate. Data in the open-field experiment were analysed using Friedman analysis of variance. P<0.05 was considered to be statistically significant.
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Results |
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The MAC and RRF values are given in Table 1. The baseline isoflurane MAC values showed a statistically significant difference for WT and KO mice (P=0.031). 7-NI pre-treatment reduced MAC in both WT and KO animals (P<0.04). Reversibility of the response to tail clamping with L-arginine was seen only in the WT animals. The RRF times were similar in both WT and KO mice. 7-NI pre-treatment significantly reduced the RRF in both WT and KO animals (both P<0.04). Reversibility of 7-NI pre-treatment with L-arginine was not seen in either WT or KO animals.
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The results of the open-field studies are shown in Figure 2. Injection of 7-NI resulted in a marked decrease in the number of line crossings in both WT and KO mice (both P<0.001). This remained low over the next 6 h (both P<0.0001). There was no difference between WT and KO mice.
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After 7-NI injection, rearing was not observed at all in the WT mice at any time during the 6 h and a median of only one rearing was observed in the KO mice (P<0.001 in each case vs control animals). There was no difference between WT and KO mice except before injection of 7-NI when the number of rearings was significantly lower in the KO animals (P=0.031). All animals had maintained their RRF throughout. There was no change in excretions in either group during the study period (data not shown).
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Discussion |
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We have shown that acute inhibition of nNOS using 7-NI reduced the MAC for isoflurane in both WT and KO animals by 29 and 38%, respectively. This effect could be reversed by co-administration of L-arginine in WT but not in KO animals. Subsequent testing for the regaining of the RRF revealed a reduction in MAC by 69% in WT and 37% in KO animals. This effect was not reversed by L-arginine.
It has previously been reported that the non-isoform specific NOS inhibitor L-NAME caused a dose-dependent and reversible reduction in the anaesthetic requirements for halothane and isoflurane in rats. However, a ceiling effect at a dose of 20 mg kg–1 L-NAME limited this effect to a reduction in MAC of up to 51% for halothane and 35% for isoflurane.1 3 4 The effect of L-NAME on MAC was disputed by another group using a similar experimental design and identical doses and volumes of L-NAME.14 15
L-Arginine analogues such as L-NAME are non-isoform specific competitive inhibitors of NOS. The inhibition is usually reversible with an excess of the NOS substrate L-arginine. The non-isoform specificity of these inhibitors leads to peripheral vasoconstriction with subsequent increases in blood pressure. The haemodynamic effects and the non-isoform specificity and possible interactions with other receptors make L-NAME a less suitable candidate for studies into the effects of nNOS inhibition in nNOS deficient mice. 7-NI structurally resembles imidazole and is thought to inhibit NOS competitively on the L-arginine binding site and non-competitively on the tetrahydrobiopterin site within the haem domain.16 17 Although 7-NI is strictly speaking not isoform specific for nNOS in in vitro studies,18 7-NI appears to have acceptable isoform specificity for nNOS possibly because of a preferred uptake into neuronal cells in in vivo studies, which may also explain the absence of haemodynamic effects.19 20 Although noting the limitations of 7-NI as a nNOS selective inhibitor for in vitro studies, we chose to use 7-NI in the absence of a well studied alternative in vivo inhibitor.
The nNOS specific inhibitor, 7-NI, has been used for in vivo experiments and a dose-dependent reduction of MAC for halothane and isoflurane in rats and sevoflurane in mice has been described.3 5–7 7-NI was given as an i.p. injection and no changes in blood pressure were reported when compared with control animals. The highest dose of 7-NI (1000 mg kg–1) reduced the MAC for halothane by 90–95% when compared with untreated controls.6 The chosen route and dose of 7-NI in our study was based on previous reports and was used at a concentration of 120 mg kg–1. 7-NI was the active compound as the vehicle, arachis oil, did not change MAC or RRF in these studies.3 5 7 Our results are in complete agreement with the previously reported studies in WT mice. Potentially, the second injection of L-arginine dissolved in sterile water could have influenced MAC determination in KO and WT mice. We therefore, elected to use untreated animals as controls for the determination of MAC and RRF.
The major stimulus for NO production in the brain is from nNOS through activation of NMDA receptors that are highly permeable to Ca2+. The N-terminal region of nNOS contains a PDZ domain, this targets nNOS to postsynaptic density protein 95 and is thought to mediate coupling of NMDA receptor activity.21 This close coupling allows tight regulation of Ca2+ influx and regulation of nNOS activity. Exon 2 of the nNOS gene encodes the PDZ domain of nNOS, which is missing in the mRNA splice variants; NOSß and nNOS
. The ß and splice variants are, therefore, not in close proximity to the NMDA receptor and occur within the cytosolic fraction of brain homogenates. Both splice variants possess catalytic activity and nNOSß is 2- to 3-fold up-regulated in KO when compared with WT mice.8 9
Although acute administration of NOS inhibitors reduces anaesthetic requirements in most studies, targeted disruption of the nNOS gene in mice (KO) resulting in the complete absence of nNOS activity, did not modify the MAC of isoflurane when compared with WT mice (1.24% vs 1.24%).4 The i.p. injection of L-NAME (10–100 mg kg–1) failed to reduce the isoflurane MAC in KO mice but reduced the MAC in WT by a maximum of 0.35% inspired isoflurane concentration when L-NAME was administered at 25 mg kg–1.
In contrast to the findings of Ichinose and colleagues,4 but similar to the results in WT mice, acute administration of nNOS specific inhibitor, 7-NI, reduced the isoflurane MAC in KO animals by 38% and the RRF by 28%. The baseline MAC for isoflurane in WT and KO mice was higher than in the previously reported study,4 and was significantly different between the WT and KO animals. Elevated MAC values between studies can be attributed to experimental variation and inter-observer variability whereas the differences between WT and KO animals represent a significantly lower sensitivity to isoflurane in KO animals than previously reported. Mouse strain may modestly influence the MAC for volatile agents. The isoflurane MAC for WT mice is reported to be between 1.3% in C57BL/6J and 1.4% in 129/SvJ mice.22 The WT animals used are the F2 hybrid of the strains C57BL/6J and 129S1/SvImJ on which the KO animals were originally derived. These WT animals are different to the control animals used by Ichinose and colleagues4 and it is conceivable that the difference between the WT and KO animals is a result of using a different control strain and represents a major limitation of this latter study.
The differences observed in isoflurane MAC for 7-NI pre-treated KO animals could be at least partially explained by the use of the specific nNOS inhibitor instead of the non-isoform specific L-NAME. Although deficient in the major neuronal isoform, nNOS, KO animals possess a residual NOS activity originally attributed to other NOS isoforms or up-regulated nNOS splice variants such as nNOS and nNOSß. This suggests either that only minimal nNOS activity is required to maintain cellular homoeostasis, or that alternative compensatory pathways exist.
The reduced isoflurane MAC resulting from 7-NI administration and the involvement of the NMDA–nitric oxide–cyclic GMP pathway was confirmed by the co-administration of the NOS substrate, L-arginine, which restored the isoflurane MAC values in WT animals. The dose of 600 mg kg–1 L-arginine was previously shown to be sufficient to reverse 7-NI inhibition of nNOS4 5 but the effects of higher doses in KO mice will need to be tested in future experiments. On a similar note the failure to reverse the effects of 7-NI in the isoflurane RRF in both the WT and KO animals are likely because of the pharmacokinetic properties and metabolism of L-arginine after 3–4 h and will require further studies. The observed values for the isoflurane RRF of 0.53% and 0.63% for WT and KO animals, respectively, are comparable to the baseline isoflurane RRF values reported4 despite the determination of the isoflurane RRF after the isoflurane MAC experiments.
Initial subjective observation after i.p. administration of 7-NI indicated a decrease in spontaneous motor activity in both WT and KO mice. A simple open-field experiment was devised to quantify this effect. A significant decrease in line crossings and number of rearings was seen in both WT and KO mice after 7-NI injection, an effect which persisted for more than 6 h. WT mice tended to be more active than the KO mice, but this effect only reached significance in the number of rearings. 7-NI has previously been reported to be an anxiolytic and sedative agent and producing motor deficits in mice utilizing a battery of neuro-behavioural tests.11 12 7-NI produced a dose-dependent decrease in locomotor activity in the open-field study measured within 1 h of i.p. administration of 7-NI in mice, whereas injection with the vehicle arachis oil had no effect.11 Arachis oil has previously been reported to be inert when used as a control in experiments using 7-NI.3 5 7 11 12 It is conceivable, that pain after the i.p. injection could potentially influence spontaneous motor activity in these mice but given that the reduction of spontaneous movements persisted for more than the studied 6 h it is unlikely that pain played a significant role. Small doses of 7-NI (10 mg kg–1) resulted in a measurable reduction in anxiety and motor activity and it could be speculated that the difference found between KO and WT mice could be because of a decrease in anxiety in these mice. However, a formal evaluation of 7-NI in KO mice would require a number of ‘state’ anxiety tests to be carried out and the creation of a dose–response curve for 7-NI.23 24 The reduction of spontaneous motor activity was evident within 1 h of i.p. administration of 7-NI and lasted for more than 6 h. However, the motor deficits appear to be reversible within 24 h after administration of 7-NI.12 The susceptibility of KO mice to 7-NI suggests that sufficient nitric oxide is available to regulate intracellular processes. This nitric oxide is most likely produced by nNOS mRNA splice variants or conceivably through endothelial NOS with subsequent diffusion of nitric oxide into neuronal cells and warrants further investigation. The comparable number of excretions throughout the experiment indicates a sufficient hydration status of the mice.
In summary, we have shown that the nNOS specific inhibitor 7-NI, reduced isoflurane MAC and spontaneous motor activity in WT and nNOS KO mice. Our results indicate that the NMDA receptor–nitric oxide–cyclic GMP pathway still remains a credible target in modulating the effects of the volatile anaesthetic agent isoflurane.
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Acknowledgments |
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We acknowledge financial support from the Royal College of Anaesthetists, the Association of Anaesthetists of Great Britain and Ireland and the Chief Scientist Office in Scotland.
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References |
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1 Johns RA, Moscicki JC, DiFazio CA. Nitric oxide synthase inhibitor dose-dependently and reversibly reduces the threshold for halothane anesthesia. A role for nitric oxide in mediating consciousness? Anesthesiology 1992; 77: 779–84[CrossRef][Web of Science][Medline]
2 Sonner JM, Antognini JF, Dutton RC, et al. Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg 2003; 97: 718–40
3 Pajewski TN, DiFazio CA, Moscicki JC, Johns RA. Nitric oxide synthase inhibitors, 7-nitro indazole and nitroG-L-arginine methyl ester, dose dependently reduce the threshold for isoflurane anesthesia. Anesthesiology 1996; 85: 1111–19[CrossRef][Web of Science][Medline]
4 Ichinose F, Huang PL, Zapol WM. Effects of targeted neuronal nitric oxide synthase gene disruption and nitroG-L-arginine methylester on the threshold for isoflurane anesthesia. Anesthesiology 1995; 83: 101–8[CrossRef][Web of Science][Medline]
5 Ichinose F, Mi WD, Miyazaki M, Onouchi T, Goto T, Morita S. Lack of correlation between the reduction of sevoflurane MAC and the cerebellar cyclic GMP concentrations in mice treated with 7-nitroindazole. Anesthesiology 1998; 89: 143–8[Web of Science][Medline]
6 Fukuda T, Saito S, Sato S, Harukuni I, Toyooka H. Halothane minimum alveolar anesthetic concentration and neuronal nitric oxide synthase activity of the dorsal horn and the locus ceruleus in rats. Anesth Analg 1999; 89: 1035–9
7 Kobayashi S, Katoh T, Iwamoto T, Bito H, Sato S. Effect of the neuronal nitric oxide synthase inhibitor 7-nitroindazole on the righting reflex ED50 and minimum alveolar concentration during sevoflurane anaesthesia in rats. Eur J Anaesthesiol 2003; 20: 212–19[CrossRef][Medline]
8 Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. Targeted disruption of the neuronal nitric oxide synthase gene. Cell 1993; 75: 1273–86[CrossRef][Web of Science][Medline]
9 Eliasson MJL, Blackshaw S, Schell MJ, Snyder SH. Neuronal nitric oxide synthase alternatively spliced forms: prominent functional localizations in the brain. Proc Natl Acad Sci USA 1997; 94: 3396–401
10 Putzke J, Seidel B, Huang PL, Wolf G. Differential expression of alternatively spliced isoforms of neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptors (NMDAR) in knockout mice deficient in nNOS (nNOS
/ mice). Mol Brain Res 2000; 85: 13–23[Medline]
11 Volke V, Soosaar A, Koks S, Bourin M, Mannisto PT, Vasar E. 7-Nitroindazole, a nitric oxide synthase inhibitor, has anxiolytic-like properties in exploratory models of anxiety. Psychopharmacology (Berl) 1997; 131: 399–405[CrossRef][Medline]
12 Araki T, Mizutani H, Matsubara M, Imai Y, Mizugaki M, Itoyama Y. Nitric oxide synthase inhibitors cause motor deficits in mice. Eur Neuropsychopharmacol 2001; 11: 125–33[Medline]
13 Crawley JN. Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res 1999; 835: 18–26[CrossRef][Web of Science][Medline]
14 Adachi T, Kurata J, Nakao S, et al. Nitric oxide synthase inhibitor does not reduce minimum alveolar anesthetic concentration of halothane in rats. Anesth Analg 1994; 78: 1154–7
15 Adachi T, Shinomura T, Nakao S, et al. Chronic treatment with nitric oxide synthase (NOS) inhibitor profoundly reduces cerebellar NOS activity, cyclic guanosine monophosphate but does not modify minimum alveolar anesthetic concentration. Anesth Analg 1995; 81: 862–5[Abstract]
16 Klatt P, Schmidt K, Brunner F, Mayer B. Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation. J Biol Chem 1994; 269: 1674–80
17 Klatt P, Schmid M, Leopold E, Schmidt K, Werner ER, Mayer B. The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. J Biol Chem 1994; 269: 13861–6
18 Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthase: structure, function and inhibition. Biochem J 2001; 357: 593–615[CrossRef][Web of Science][Medline]
19 Moore PK, Babbedge RC, Wallace P, Gaffen ZA, Hart SL. 7-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure. Br J Pharmacol 1993; 108: 296–7[Web of Science][Medline]
20 Yoshida T, Limmroth V, Irikura K, Moskowitz MA. The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels. J Cereb Blood Flow Metab 1994; 14: 924–9[Web of Science][Medline]
21 Barañano DE, Snyder SH. Neural roles for heme oxygenase: contrast to nitric oxide synthase. Proc Natl Acad Sci USA 2001; 98: 10996–1002
22 Sonner JM, Gong D, Eger EI II. Naturally occurring variability in anesthetic potency among inbred mouse strains. Anesth Analg 2000; 91: 720–6
23 Hascoet M, Bourin M. A new approach to the light/dark test procedure in mice. Pharmacol Biochem Behav 1998; 60: 645–53[CrossRef][Web of Science][Medline]
24 Belzung C, Griebel G. Measuring normal and pathological anxiety-like behaviour in mice: a review. Behav Brain Res 2001; 125: 141–9[CrossRef][Web of Science][Medline]
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