- Email: [email protected]
Antisense knockdown of neuronal nitric oxide synthase antagonizes nitrous oxide-induced behavior
Brain Research 968 (2003) 167–170 www.elsevier.com / locate / brainres
Short communication
Antisense knockdown of neuronal nitric oxide synthase antagonizes nitrous oxide-induced behavior Shuang Li a,b , Yang Dai d , Raymond M. Quock a,b,c , * a
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, P.O. Box 646534, Pullman, WA 99164 -6534, USA b Graduate Program in Pharmacology and Toxicology, Washington State University, Pullman, WA, USA c Center for Integrated Biotechnology, Washington State University, Pullman, WA, USA d Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, USA Accepted 6 January 2003
Abstract The behavioral effects of nitrous oxide (N 2 O) were antagonized by non-specific inhibitors of nitric oxide synthase (NOS). To identify the isoform of NOS involved in this response, mice were pretreated with an antisense oligodeoxynucleotide (AS-ODN) against neuronal NOS, and then tested in a light / dark exploration paradigm. The AS-ODN but not the mismatch ODN significantly antagonized N 2 O induced behavior and also reduced NOS activity in the cerebellum and hippocampus. These results implicate neuronal NOS in the N 2 O response. 2003 Elsevier Science B.V. All rights reserved. Theme: Neurotransmsitters, modulators, transporters and receptors Topic: Behavioral pharmacology Keywords: Nitrous oxide; Nitric oxide; Antisense; Anxiolytic effect
Earlier experiments from our laboratory have demonstrated an essential role of nitric oxide (NO) in mediating the anxiolytic effects of N 2 O and benzodiazepines [2,8,9]. There are three different isoforms of NOS (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) but it is most likely that nNOS would be involved in mediation of a behavioral drug response. To confirm this suspicion, we employed an AS-ODN knockdown strategy, which will be highly selective for arresting nNOS mRNA translation into functional enzyme. The regional brain NOS activity was quantified to confirm that the AS-ODN did reduce NOS activity. Male NIH Swiss mice, weighing 18–22 g, were tested for their behavioral response to N 2 O following pretreatment with nNOS AS- or mismatch (MM)-ODNs, which were purchased from Molecula Research Laboratories (Herndon, Virginia). The AS-ODN sequence (59-GAA *Corresponding author. Tel.: 11-509-335-5956; fax: 11-509-3355902. E-mail address: [email protected] (R.M. Quock).
TCC TCT CCC CGC CCA-39) was 18-mer (18 bases) and was designed to flank exon 18 within the mouse nNOS mRNA (nt 2815–2833, GenBank accession No D14552) [7]. The corresponding MM-ODN had the sequence 59GAA TCT CCT CCC GCC CCA-39. Both AS- and MMODNs were phosphorothioates to prevent degradation by endonucleases. All were purified by RP-HPLC and dissolved in 0.9% saline before injection (25 mg / 4 ml). On days 1, 3 and 5, mice received single intracerebroventricular (i.c.v.) injections of saline or an ODN (25 mg / 4 ml per injection) on alternating sides of the brain (i.e., day 1, left side; day 3, right side; and day 5, left side). Briefly, mice were lightly anesthetized with halothane, an incision was made in the scalp, and the skin was pulled back to expose the calvarium. The injection was made freehand into the lateral cerebral ventricle using a 26gauge microsyringe at a point on the calvarium 1.0 mm lateral to and 1.0 mm caudal to bregma to a depth of 2.5 mm from the skull surface. On day 6, mice were tested in a light / dark exploration box (45 cm L327 cm W327 cm H) with a 75375-mm
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02289-3
168
S. Li et al. / Brain Research 968 (2003) 167–170
aperture at floor level between the light and dark compartments [8]. N 2 O, U.S.P. and O 2 , U.S.P. (A&L Welding, Spokane, Washington) were delivered into the light / dark exploration box using a standard dental sedation system (Porter, Hatfield, PA). The concentration of N 2 O and O 2 delivered into the box was monitored using a POET II anesthetic monitoring system (Criticare, Milwaukee, WI). Mice were individually placed in the center of the light compartment of the box, which had been pre-filled with 50% N 2 O and 50% O 2 , facing away from the divider. The total exposure time of 5 min was coincident with the 5-min observation period. The time spent in the light compartment of the box as well as the number of transitions between the light and dark compartments were recorded for each mouse. A mouse was considered to have entered the new area when all four legs crossed the threshold into the compartment. Score was done by direct observation by the same individual. In preliminary experiments, the difference between AS- and missense-pretreated groups was so marked that there was a high degree of agreement between blind and non-blind raters. To determine the degree of inhibition of NOS expression, different but similarly pretreated groups of mice were euthanized on day 6. Their brains were quickly removed, and then the cerebella, hippocampi and amygdalas were dissected out on an ice-cold glass Petri dish. Samples were immediately frozen in liquid nitrogen and stored at 280 8C until the analysis. NOS activity was assayed by measuring the Ca 21 -dependent conversion of [ 14 C]L-arginine to [ 14 C]L-citrulline [6]. Briefly, on the day of the assay, tissue samples were homogenized in 10 vol (w / v) of 50 mM Tris–HCl (pH 7.4) buffer containing 1 mM EDTA and 1 mM EGTA. After centrifugation (12 000 rpm for 20 min at
4 8C), 20 ml of supernatant were added to 40 ml of 50 mM Tris–HCl (pH 7.4) buffer containing 1.0 mM NADPH, 3.0 mM BH 4 , 1.0 mM FAD 1.0 mM FMN, 1.25 mM CaCl 2 , and 1.25 mCi ml 21 [ 14 C]L-arginine (specific activity, 348 mCi / mmol; Amersham Biosciences, Piscataway, NJ) and incubated for 30 min at 37 8C. The reaction was terminated by addition of 400 ml stop buffer containing 50 mM Hepes (pH 5.5) and 5 mM EDTA. Then the reaction mixture was applied onto a chromatographic column containing 40 mg of Dowex AG50WX-8 resin for separation of [ 14 C]Lcitrulline from the unreacted [ 14 C]L-arginine by cationexchange chromatography and collected into a scintillation vial. Thereafter, the samples were counted for the amount of radioactivity using a model A2500 liquid scintillation counter (Packard Instrument, Meriden, CT). The protein content of the supernatant was determined by the bicinchoninic acid method using bovine albumin as standard [10] with a commercially available assay kit (Pierce Chemical, Rockford, IL). NOS enzyme activity was expressed in terms of pmol citrulline formed / mg protein per min. All data were analyzed by one-way analyses of variance (ANOVA). When a significant F value was found, post-hoc analysis was performed by the Newman–Keuls multiplecomparison test. Mice exposed to 50% N 2 O exhibited increases in both time spent in the light compartment as well as number of transitions between compartments (Fig. 1A,B). These increases were all statistically significant from the baseline behavior exhibited by control (room air-exposed) animals (P,0.01). Compared to a saline pretreatment group, pretreatment with AS-ODN antagonized the N 2 O-induced increases in both time spent in the light compartment and
Fig. 1. Influence of AS- and MM-ODNs on 50% N 2 O-induced changes in (A) time spent in the light compartment and (B) number of transitions. The data are expressed as mean6S.E.M. of 12–13 mice per group. Significance of difference: *P,0.05, and **P,0.01, compared to room air (RA)1saline; &P,0.03, and &&P,0.01, compared to N 2 O1saline (one-way ANOVA and Newman–Keuls post-hoc test).
S. Li et al. / Brain Research 968 (2003) 167–170
169
Fig. 2. Effect of AS- and MM-ODNs on regional brain NOS activity. The data are expressed as mean6S.E.M. of nine to 11 mice per group. Significance of difference: **P,0.01, ***P,0.001, compared with saline control (one-way ANOVA and Newman–Keuls post-hoc test).
transition number (P,0.05 or P,0.01); the MM-ODN pretreatment group was not different from the saline pretreatment group. Not only did the AS-ODN pretreatment significantly antagonize the N 2 O-induced behavioral effect, but it also significantly reduced levels of NOS activity in both cerebellum and hippocampus (P,0.01 or P,0.001) though not in the amygdala (Fig. 2). Pretreatment with MM-ODN had no such effect on regional brain NOS activity. Inhibition of a physiological or pharmacological function by competitive inhibition of NOS has been the most widely employed criterion for implicating NO in that function. Earlier studies in our laboratory have utilized G L-arginine-derived analogs such as L-NOARG (N -L-nitro G arginine), L-NAME (N -nitro-L-arginine methylester hydrochloride) or L-NMMA (N G -L-nitromonomethyl arginine), which are all considered to be non-selective inhibitors of NOS. We have also used 7-nitroindazole (7-NI), which is ostensibly selective for nNOS; however, there are reports in the literature that 7-NI may actually not be specific for nNOS and can also inhibit eNOS and iNOS [1]. In the present study, an AS-ODN strategy was adopted to take advantage of the high degree of selectivity of AS-ODNs for the targeted mRNA. The AS-ODN used in this study was specific for an 18-base section flanking exon 18 within the mouse nNOS mRNA [7]. Pretreatment with this AS-ODN significantly antagonized N 2 O-induced behavior and markedly reduced NOS activity in the cerebellum and hippocampus. The inactivity of the MM-ODN in duplicating the AS-ODN effects confirms that specific inhibition of nNOS expression underlies antagonism of the N 2 O responses. The
cerebellum is the brain area in which NOS is most richly expressed [3], while the hippocampus and amygdala are brain regions that have been implicated in the regulation of anxiety [4,5]. These results suggest that NOS activity in the hippocampus may play a critical role in mediating the anxiolytic-like behavioral response to N 2 O.
Acknowledgements This work was support by NIH Grant DA-10343.
References [1] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure, function and inhibition, Biochem. J. 357 (2001) 593–615. [2] P.W. Caton, S.A. Tousman, R.M. Quock, Involvement of nitric oxide anxiolysis in the elevated plus-maze, Pharmacol. Biochem. Behav. 48 (1994) 689–692. [3] N. Clavier, J.R. Tobin, J.R. Kirsch, M. Izuta, R.J. Traystman, Brain nitric oxide synthase activity in normal, hypertensive, and strokeprone rats, Stroke 25 (1994) 1674–1678. [4] G.W. Dent, D.M. O’Dell, J.H. Eberwine, Gene expression profiling in the amygdala: an approach to examine the molecular substrates of mammalian behavior, Physiol. Behav. 73 (2001) 841–847. [5] M. Fendt, M.S. Fanselow, The neuroanatomical and neurochemical basis of conditioned fear, Neurosci. Biobehav. Rev. 23 (1999) 743–760. [6] P.L. Huang, T.M. Dawson, D.S. Bredt, S.H. Snyder, M.C. Fishman, Targeted disruption of the neuronal nitric oxide synthase gene, Cell 75 (1993) 1273–1286. [7] Y.A. Kolesnikov, Y.X. Pan, A.M. Babey, S. Jain, R. Wilson, G.W. Pasternak, Functionally differentiating two neuronal nitric oxide synthase isoforms through antisense mapping: evidence for opposing
170
S. Li et al. / Brain Research 968 (2003) 167–170
NO actions on morphine analgesia and tolerance, Proc. Natl. Acad. Sci. USA 94 (1997) 8220–8225. [8] S. Li, R.M. Quock, Comparison of chlordiazepoxide and N 2 Oinduced behaviors in the light / dark exploration test, Pharmacol. Biochem. Behav. 68 (2001) 789–796. [9] R.M. Quock, E. Nguyen, Possible involvement of nitric oxide in
chlordiazepoxide-induced anxiolysis in mice, Life Sci. 51 (1992) 255–260. [10] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85.
Short communication
Antisense knockdown of neuronal nitric oxide synthase antagonizes nitrous oxide-induced behavior Shuang Li a,b , Yang Dai d , Raymond M. Quock a,b,c , * a
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, P.O. Box 646534, Pullman, WA 99164 -6534, USA b Graduate Program in Pharmacology and Toxicology, Washington State University, Pullman, WA, USA c Center for Integrated Biotechnology, Washington State University, Pullman, WA, USA d Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, USA Accepted 6 January 2003
Abstract The behavioral effects of nitrous oxide (N 2 O) were antagonized by non-specific inhibitors of nitric oxide synthase (NOS). To identify the isoform of NOS involved in this response, mice were pretreated with an antisense oligodeoxynucleotide (AS-ODN) against neuronal NOS, and then tested in a light / dark exploration paradigm. The AS-ODN but not the mismatch ODN significantly antagonized N 2 O induced behavior and also reduced NOS activity in the cerebellum and hippocampus. These results implicate neuronal NOS in the N 2 O response. 2003 Elsevier Science B.V. All rights reserved. Theme: Neurotransmsitters, modulators, transporters and receptors Topic: Behavioral pharmacology Keywords: Nitrous oxide; Nitric oxide; Antisense; Anxiolytic effect
Earlier experiments from our laboratory have demonstrated an essential role of nitric oxide (NO) in mediating the anxiolytic effects of N 2 O and benzodiazepines [2,8,9]. There are three different isoforms of NOS (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) but it is most likely that nNOS would be involved in mediation of a behavioral drug response. To confirm this suspicion, we employed an AS-ODN knockdown strategy, which will be highly selective for arresting nNOS mRNA translation into functional enzyme. The regional brain NOS activity was quantified to confirm that the AS-ODN did reduce NOS activity. Male NIH Swiss mice, weighing 18–22 g, were tested for their behavioral response to N 2 O following pretreatment with nNOS AS- or mismatch (MM)-ODNs, which were purchased from Molecula Research Laboratories (Herndon, Virginia). The AS-ODN sequence (59-GAA *Corresponding author. Tel.: 11-509-335-5956; fax: 11-509-3355902. E-mail address: [email protected] (R.M. Quock).
TCC TCT CCC CGC CCA-39) was 18-mer (18 bases) and was designed to flank exon 18 within the mouse nNOS mRNA (nt 2815–2833, GenBank accession No D14552) [7]. The corresponding MM-ODN had the sequence 59GAA TCT CCT CCC GCC CCA-39. Both AS- and MMODNs were phosphorothioates to prevent degradation by endonucleases. All were purified by RP-HPLC and dissolved in 0.9% saline before injection (25 mg / 4 ml). On days 1, 3 and 5, mice received single intracerebroventricular (i.c.v.) injections of saline or an ODN (25 mg / 4 ml per injection) on alternating sides of the brain (i.e., day 1, left side; day 3, right side; and day 5, left side). Briefly, mice were lightly anesthetized with halothane, an incision was made in the scalp, and the skin was pulled back to expose the calvarium. The injection was made freehand into the lateral cerebral ventricle using a 26gauge microsyringe at a point on the calvarium 1.0 mm lateral to and 1.0 mm caudal to bregma to a depth of 2.5 mm from the skull surface. On day 6, mice were tested in a light / dark exploration box (45 cm L327 cm W327 cm H) with a 75375-mm
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02289-3
168
S. Li et al. / Brain Research 968 (2003) 167–170
aperture at floor level between the light and dark compartments [8]. N 2 O, U.S.P. and O 2 , U.S.P. (A&L Welding, Spokane, Washington) were delivered into the light / dark exploration box using a standard dental sedation system (Porter, Hatfield, PA). The concentration of N 2 O and O 2 delivered into the box was monitored using a POET II anesthetic monitoring system (Criticare, Milwaukee, WI). Mice were individually placed in the center of the light compartment of the box, which had been pre-filled with 50% N 2 O and 50% O 2 , facing away from the divider. The total exposure time of 5 min was coincident with the 5-min observation period. The time spent in the light compartment of the box as well as the number of transitions between the light and dark compartments were recorded for each mouse. A mouse was considered to have entered the new area when all four legs crossed the threshold into the compartment. Score was done by direct observation by the same individual. In preliminary experiments, the difference between AS- and missense-pretreated groups was so marked that there was a high degree of agreement between blind and non-blind raters. To determine the degree of inhibition of NOS expression, different but similarly pretreated groups of mice were euthanized on day 6. Their brains were quickly removed, and then the cerebella, hippocampi and amygdalas were dissected out on an ice-cold glass Petri dish. Samples were immediately frozen in liquid nitrogen and stored at 280 8C until the analysis. NOS activity was assayed by measuring the Ca 21 -dependent conversion of [ 14 C]L-arginine to [ 14 C]L-citrulline [6]. Briefly, on the day of the assay, tissue samples were homogenized in 10 vol (w / v) of 50 mM Tris–HCl (pH 7.4) buffer containing 1 mM EDTA and 1 mM EGTA. After centrifugation (12 000 rpm for 20 min at
4 8C), 20 ml of supernatant were added to 40 ml of 50 mM Tris–HCl (pH 7.4) buffer containing 1.0 mM NADPH, 3.0 mM BH 4 , 1.0 mM FAD 1.0 mM FMN, 1.25 mM CaCl 2 , and 1.25 mCi ml 21 [ 14 C]L-arginine (specific activity, 348 mCi / mmol; Amersham Biosciences, Piscataway, NJ) and incubated for 30 min at 37 8C. The reaction was terminated by addition of 400 ml stop buffer containing 50 mM Hepes (pH 5.5) and 5 mM EDTA. Then the reaction mixture was applied onto a chromatographic column containing 40 mg of Dowex AG50WX-8 resin for separation of [ 14 C]Lcitrulline from the unreacted [ 14 C]L-arginine by cationexchange chromatography and collected into a scintillation vial. Thereafter, the samples were counted for the amount of radioactivity using a model A2500 liquid scintillation counter (Packard Instrument, Meriden, CT). The protein content of the supernatant was determined by the bicinchoninic acid method using bovine albumin as standard [10] with a commercially available assay kit (Pierce Chemical, Rockford, IL). NOS enzyme activity was expressed in terms of pmol citrulline formed / mg protein per min. All data were analyzed by one-way analyses of variance (ANOVA). When a significant F value was found, post-hoc analysis was performed by the Newman–Keuls multiplecomparison test. Mice exposed to 50% N 2 O exhibited increases in both time spent in the light compartment as well as number of transitions between compartments (Fig. 1A,B). These increases were all statistically significant from the baseline behavior exhibited by control (room air-exposed) animals (P,0.01). Compared to a saline pretreatment group, pretreatment with AS-ODN antagonized the N 2 O-induced increases in both time spent in the light compartment and
Fig. 1. Influence of AS- and MM-ODNs on 50% N 2 O-induced changes in (A) time spent in the light compartment and (B) number of transitions. The data are expressed as mean6S.E.M. of 12–13 mice per group. Significance of difference: *P,0.05, and **P,0.01, compared to room air (RA)1saline; &P,0.03, and &&P,0.01, compared to N 2 O1saline (one-way ANOVA and Newman–Keuls post-hoc test).
S. Li et al. / Brain Research 968 (2003) 167–170
169
Fig. 2. Effect of AS- and MM-ODNs on regional brain NOS activity. The data are expressed as mean6S.E.M. of nine to 11 mice per group. Significance of difference: **P,0.01, ***P,0.001, compared with saline control (one-way ANOVA and Newman–Keuls post-hoc test).
transition number (P,0.05 or P,0.01); the MM-ODN pretreatment group was not different from the saline pretreatment group. Not only did the AS-ODN pretreatment significantly antagonize the N 2 O-induced behavioral effect, but it also significantly reduced levels of NOS activity in both cerebellum and hippocampus (P,0.01 or P,0.001) though not in the amygdala (Fig. 2). Pretreatment with MM-ODN had no such effect on regional brain NOS activity. Inhibition of a physiological or pharmacological function by competitive inhibition of NOS has been the most widely employed criterion for implicating NO in that function. Earlier studies in our laboratory have utilized G L-arginine-derived analogs such as L-NOARG (N -L-nitro G arginine), L-NAME (N -nitro-L-arginine methylester hydrochloride) or L-NMMA (N G -L-nitromonomethyl arginine), which are all considered to be non-selective inhibitors of NOS. We have also used 7-nitroindazole (7-NI), which is ostensibly selective for nNOS; however, there are reports in the literature that 7-NI may actually not be specific for nNOS and can also inhibit eNOS and iNOS [1]. In the present study, an AS-ODN strategy was adopted to take advantage of the high degree of selectivity of AS-ODNs for the targeted mRNA. The AS-ODN used in this study was specific for an 18-base section flanking exon 18 within the mouse nNOS mRNA [7]. Pretreatment with this AS-ODN significantly antagonized N 2 O-induced behavior and markedly reduced NOS activity in the cerebellum and hippocampus. The inactivity of the MM-ODN in duplicating the AS-ODN effects confirms that specific inhibition of nNOS expression underlies antagonism of the N 2 O responses. The
cerebellum is the brain area in which NOS is most richly expressed [3], while the hippocampus and amygdala are brain regions that have been implicated in the regulation of anxiety [4,5]. These results suggest that NOS activity in the hippocampus may play a critical role in mediating the anxiolytic-like behavioral response to N 2 O.
Acknowledgements This work was support by NIH Grant DA-10343.
References [1] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure, function and inhibition, Biochem. J. 357 (2001) 593–615. [2] P.W. Caton, S.A. Tousman, R.M. Quock, Involvement of nitric oxide anxiolysis in the elevated plus-maze, Pharmacol. Biochem. Behav. 48 (1994) 689–692. [3] N. Clavier, J.R. Tobin, J.R. Kirsch, M. Izuta, R.J. Traystman, Brain nitric oxide synthase activity in normal, hypertensive, and strokeprone rats, Stroke 25 (1994) 1674–1678. [4] G.W. Dent, D.M. O’Dell, J.H. Eberwine, Gene expression profiling in the amygdala: an approach to examine the molecular substrates of mammalian behavior, Physiol. Behav. 73 (2001) 841–847. [5] M. Fendt, M.S. Fanselow, The neuroanatomical and neurochemical basis of conditioned fear, Neurosci. Biobehav. Rev. 23 (1999) 743–760. [6] P.L. Huang, T.M. Dawson, D.S. Bredt, S.H. Snyder, M.C. Fishman, Targeted disruption of the neuronal nitric oxide synthase gene, Cell 75 (1993) 1273–1286. [7] Y.A. Kolesnikov, Y.X. Pan, A.M. Babey, S. Jain, R. Wilson, G.W. Pasternak, Functionally differentiating two neuronal nitric oxide synthase isoforms through antisense mapping: evidence for opposing
170
S. Li et al. / Brain Research 968 (2003) 167–170
NO actions on morphine analgesia and tolerance, Proc. Natl. Acad. Sci. USA 94 (1997) 8220–8225. [8] S. Li, R.M. Quock, Comparison of chlordiazepoxide and N 2 Oinduced behaviors in the light / dark exploration test, Pharmacol. Biochem. Behav. 68 (2001) 789–796. [9] R.M. Quock, E. Nguyen, Possible involvement of nitric oxide in
chlordiazepoxide-induced anxiolysis in mice, Life Sci. 51 (1992) 255–260. [10] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85.