Putative anxiety-linked effects of the nitric oxide synthase inhibitor l-NAME in three murine exploratory behavior models

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Abstract

The aim of the current study was to extend investigation into possible linkage between nitric oxide (NO) and anxiety-linked behavior using a battery of tests. Effects of the NO synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME) were investigated in three murine models of anxiety—the light–dark, hole-board and elevated plus-maze—in between-groups designs. Treatment groups included l-NAME (0 [vehicle, or Veh], 10, 25, and 50 mg/kg) and 50 mg/kg of the inactive isomer NG-nitro-d-arginine methyl ester (d-NAME) injected subcutaneously. Mice exhibited a robust anxiogenic-like response profile reflected by dose-related decreases in both light–dark (transitions and time in lighted area) and hole-board (head dips and time spent head dipping) test measures, reaching statistical significance at 25 and 50 mg/kg l-NAME when compared to Veh controls (P<.05 or .01; Dunnett's t test), while distance traveled and rearing showed no significant differential pattern in either model. In both models, there was a strong dissociation between nonspecific locomotion and putative exploratory behaviors. d-NAME was not significantly different from Veh condition in either model, indicating a stereospecific action and supporting NO involvement. A dose-related decrease was also observed for several traditional and ethological measures in the plus-maze; however, the effect was limited and relatively weak or absent; with the exception of open-arm and percent open-arm entries, putative anxiety-sensitive measures reached statistical significance only at the highest dose. Reductions in motor activity compromised ability to dissociate an anxiety linkage from a nonspecific motor effect in most measures. It is concluded that the hole-board and light–dark tests provide indication of anxiogenic-like action of NOS inhibition, suggesting that NO has an anxiolytic action. Data from the plus-maze are unclear, owing to a confounding motor influence in most measures.

Introduction

The naturally occurring vasodilating gas, nitric oxide (NO), has been implicated in the recent literature as playing an important role in a broad spectrum of physiological and behavioral functions in animals. These include learning and memory, antinociceptive action/effects, sexual functioning, a number of ingestive-linked behaviors, regulation of autonomic functions, drug dependence, and anxiety-linked behaviors (for reviews, see Krukoff, 1999, Nelson et al., 1997, Riedel and Neeck, 2001, Szabo, 1996, Uzbay and Oglesby, 2001). There is evidence that NO functions as a neurotransmitter and intracellular messenger/signal in both central and peripheral nervous systems Dawson and Snyder, 1994, Moncada et al., 1991. NO is synthesized from the amino acid l-arginine (l-arg) through action of the catalytic enzyme NO synthase (NOS) (Moncada et al., 1991).

Of particular interest to our laboratory have been a number of reports implicating NO signaling in mechanisms involved in anxiety-linked behaviors and which have also generated a number of conflicting findings. As is a common strategy in studies probing NO involvement, NO production was restricted through inhibiting NOS. Two of the earliest studies reported that acute systemic injection of the neuronal NOS inhibitor NG-nitro-l-arginine (l-NOARG) antagonized the anxiolytic effect of the benzodiazepine drug chlordiazepoxide (CP) (Quock and Nguyen, 1992) and of the anesthetic gas nitrous oxide (N2O) (Caton et al., 1994) in the elevated plus-maze in mice, thereby indicating an anxiogenic action of NOS inhibition. In both of these studies, l-NOARG's antagonist effect was reversed by intracerebroventricular administration of l-arg, the natural substrate for NOS, but not by the inactive isomer, d-arginine (d-arg), thus linking l-NOARG's influence to inhibition of NO production. More recently, an anxiogenic-like effect of NOS inhibition has also been reported in the elevated plus-maze in rat with systemic De Oliveira et al., 1997, Vale et al., 1998, Pokk and Vali, 2002 and CNS (Monzón et al., 2001) administration of l-NOARG or NG-nitro-l-arginine methyl ester (l-NAME) and in the light–dark test in mice following systemic administration of the selective neuronal NOS inhibitor 7-nitroindazole (7-NI) (Li and Quock, 2001). Most recently, it was reported that intracerebroventricular administration of an NO donor induced anxiolytic-like action in the light–dark test in mice (Li and Quock, 2002).

In contrast, several investigators have recently reported anxiolytic-like effects of l-NAME injected systemically (Faria et al., 1997) or centrally into the dorsal periaqueductal gray (PAG) (Guimarães et al., 1994) and of systemic injections of 7-NI Dunn et al., 1998, Volke et al., 1997, Yildiz et al., 2000 in the elevated plus-maze in the rat. Anxiolytic-like action of 7-NI has also been observed for the rat social interaction test, and for the mouse plus-maze and light–dark exploratory tests (Volke et al., 1997). NOS inhibition has also been reported to reduce isolation-induced ultrasonic vocalizations in rat pups Campbell et al., 1999, Podhorna and Brown, 1999. The basis(es) for these differing findings is unknown.

The present study further probed the effect of the nonselective NOS inhibitor l-NAME in the mouse light–dark exploratory test, a paradigm previously observed to reflect anxiolytic-like behavior only under quite high doses of 7-NI (Volke et al., 1997), an exploratory model in which l-NAME has not previously been evaluated—the mouse hole-board model—and the elevated plus-maze. In light of the widespread use of the plus-maze in the research herein cited, it was considered essential to include this paradigm in the battery of tests for comparison control purposes. We also included several ethological, as well as the more traditional/conventional, measures in the plus-maze—measures only recently reported in purported NO-linked studies.

Section snippets

Animals

Adult male ICR mice from our breeding colony, weighing approximately 35–50 g at time of testing, were group housed in standard 15×26×12-cm-high opaque polypropylene tub-type cages (three to five animals per cage) and maintained on a 12:12 light–dark cycle (lights on from 0700 to 1900 h) in a temperature- and humidity-controlled colony room with ad lib access to pelleted food (Teklad rodent diet 8604) and tap water. Animals were used only once, and all testing procedures were carried out during

Light–dark test

Light–dark measures are shown in Fig. 1. Both number of between-compartment transitions and time spent in lighted compartment were attenuated in a dose-related manner. The ANOVAs yielded F(4,95)=7.14 and 3.52 for transitions and time in lighted compartment, respectively (P=.0001 and .010, respectively). When compared to Veh condition, mice administered doses of either 25 or 50 mg/kg of l-NAME showed statistically significantly fewer transitions and less time in lighted area (P<.05 or .01,

Discussion

The aim of the current study was to further investigate the possible effect(s) of NOS inhibition on putative anxiety-linked behaviors, herein using a battery of three of the more widely used and validated animal models employed in probing drug effects/influence on anxiety-linked mechanisms and behavior and using a series of systemically administered moderate doses of the nonspecific NOS inhibitor l-NAME-doses often reported in the NO literature. We report findings for the hole-board test, which

Acknowledgements

The authors thank E. Buth, K. Chari, J. Galloway, T. Hutton, N. Lewis, C. McDonald, J. Pelky, S. Scudder, S. Szwed, and A. Viencz for their invaluable assistance in data collection and/or review of videotapes.

References (37)

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