Zolpidem, Triazolam, and Diazepam Decrease Distress Vocalizations in Mouse Pups: Differential Antagonism by Flumazenil and b-Carboline-3-carboxylate-t-butyl ester (b-CCt)

In response to stressful events, neonatal mice emit ultrasonic vocalizations (USVs), which are suppressed by BZ agonists. The present study examined the role of the benzodiazepine/a1 (BZ/a1) receptor subtype in the suppression engendered by the BZ/a1-preferring agonist zolpidem and the nonselective BZ agonists triazolam and diazepam. The role of BZ receptor subtypes was explored further by conducting antagonism studies using the BZ/a1-preferring antagonist b-carboline-3-carboxylate-t-butyl ester (b-CCt), in comparison with the nonselective BZ antagonist flumazenil. Mouse pups (CFW strain) were separated from their dam and littermates at day 7, and placed for 4 min in a test chamber with reduced ambient temperature (19 6 1°C) for recording USVs, motor incoordination (measured as a pup rolling on its back per grid cross), and body temperature. Zolpidem, triazolam, and diazepam suppressed USVs in a dose-dependent manner, concomitant with increases in incoordination and augmentation of hypothermia. These effects of the three BZ agonists were blocked by flumazenil in a manner consistent with surmountable antagonism. The ability of zolpidem, but not triazolam or diazepam, to suppress USVs and augment hypothermia was antagonized by b-CCt, whereas the increase in motor incoordination engendered by zolpidem, triazolam, and diazepam was not sensitive to b-CCt administration. Collectively, these results suggest that zolpidem suppresses distress USVs in mouse pups by a mechanism distinct from that of typical BZs. Furthermore, suppression of distress USVs by zolpidem may involve BZ/a1 receptors and a nonanxiolytic mechanism, such as hypothermia. Zolpidem is an imidazopyridine ligand that binds to benzodiazepine (BZ) recognition sites and is used for treatment of sleep disorders (for reviews, see Rush, 1998; Doble, 1999). The receptor binding profile of zolpidem is distinct from that of typical BZs in that it displays highest affinity at g-aminobutyric acid A receptors expressing a1 subunits (BZ/a1 receptors) but low-to-moderate affinity at receptors expressing a2, a3, and a5 subunits (Pritchett and Seeburg, 1990; Hadingham et al., 1993; Cox et al., 1995). In contrast, typical BZs (e.g., diazepam, triazolam) interact with all of the BZ sites with nearly equal affinities (for review, see Lüddens et al., 1995). The behavioral effects produced by zolpidem also appear to differ from those of typical BZs. In particular, zolpidem is relatively ineffective in tests predictive of anxiolytic activity (for review, see Sanger et al., 1994), raising the possibility that action at BZ/a1 receptors does not contribute to the anxiolytic activity of BZ ligands (cf. Rudolph et al., 1999; Löw et al., 2000; McKernan et al., 2000). In contrast to the findings with zolpidem, investigations using the relatively selective BZ/a1 antagonist b-carboline3-carboxylate-t-butyl ester (b-CCt) have implicated a role for the BZ/a1 site in mediating anxiolytic effects (Shannon et al., 1984; Belzung et al., 2000; Huang et al., 2000). For example, b-CCt antagonized the effects of diazepam in several tests of anxiolytic activity (Shannon et al., 1984; Griebel et al., 1999). The reason for this discrepancy between a BZ/a1-preferring agonist and antagonist is not clear, although b-CCt binding to a2-, a3-, or a5-containing receptors at high doses remains a possibility. An alternative explanation has been suggested based on other behavioral effects of zolpidem (Griebel et al., 1999). In this regard, zolpidem and other BZ/a1 agonists engender marked sedation and impairment of motor activity (for review, see Sanger et al., 1994). This observation has led to the hypothesis that anxiolytic-like effects of BZ/a1-selecThis research was supported by U.S. Public Health Service Grants DA11792 (to J.K.R.), MH46851 (to J.M.C.), and RR00168 (administered to the New England Regional Primate Research Center). 1 Current address: Pfizer International-La Jolla, 10777 Science Center Dr., San Diego, CA 92121. ABBREVIATIONS: BZ, benzodiazepine; b-CCt, b-carboline-3-carboxylate-t-butyl ester; USV, ultrasonic vocalization; 5-HT, 5-hydroxytryptamine; CI, confidence interval. 0022-3565/01/2971-247–253$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 297, No. 1 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3563/893786 JPET 297:247–253, 2001 Printed in U.S.A. 247 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from tive agonists are masked, or counteracted, by effects on motoric function (cf. Griebel et al., 1999). Assessment of whether BZ/a1-selective agonists produce anxiolytic effects may be facilitated by the use of procedures in which anxiolytic-related behavior can be separated from motor behavior. One such approach uses “distress” or “maternal separation” vocalizations emitted by neonatal animals when placed in contexts associated with stressful events (Winslow and Insel, 1991; Miczek et al., 1995). For example, neonatal rodents emit ultrasonic vocalizations (USVs) under conditions such as maternal separation, reduced ambient temperature, hunger, and rough handling (Okon, 1970; for review, see Miczek et al., 1995). USVs are suppressed by both BZs and anxiolytic 5-hydroxytryptamine (5-HT) agonists (Miczek et al., 1995; Olivier et al., 1998a,b; Fish et al., 2000). Suppression of USVs by anxiolytics can be dissociated from motoric impairment; for example, USV suppression by BZs and 5-HT agonists often occurs in the absence of alterations in motor activity (Olivier et al., 1998a,b; Fish et al., 2000). Accordingly, suppression of USVs in neonatal rodents may prove useful in discerning the role of BZ receptor subtypes in mediating the anxiolytic-like effects of BZ agonists. In support of this idea, several studies have shown suppression of USVs in rat pups by BZ ligands (Insel et al., 1986; Gardner and Budhram, 1987; Vivian et al., 1997; Olivier et al., 1998a). In particular, Olivier et al. (1998a) demonstrated a clear suppression of USVs in rat pups following zolpidem administration that was not accompanied by impairment of motor activity, consistent with the hypothesis that BZ/a1-selective agonists may possess anxiolytic-like effects under certain conditions. As with rats, neonatal mice emit USVs during maternal separation and cold ambient temperatures, and these USVs are suppressed by BZ agonist administration (Benton and Nastiti, 1988, 1991; Fish et al., 2000). The present study examined the ability of the BZ/a1 agonist zolpidem to suppress USVs in mouse pups, in comparison to the suppression engendered by the nonselective BZs diazepam and triazolam (Ducic et al., 1993; Hadingham et al., 1993). The role of BZ/a1 receptors was examined by conducting antagonism studies using b-CCt, in comparison with the nonselective BZ antagonist flumazenil. To evaluate the specificity of the effects of BZ ligands on USVs, the effects of these drugs on motor incoordination and thermoregulation were measured concurrently. The goal of these studies was to use USVs as a measure of anxiolytic activity not dependent on motor behavior to more clearly discern whether the BZ/a1 receptor subtype plays a fundamental role in the anxiolytic effects of typical and atypical BZ agonists. Materials and Methods Animals. Seven-day-old CFW mouse pups (N 5 450) from litters that had 7 to 13 pups were used in the experiments. They were bred on site and housed with their parents (Charles River Breeding Labs, Wilmington, MA) with free access to rodent chow (Purina, St. Louis, MO) and tap water in clear polycarbonate cages (28 3 17 3 14 cm) and pine shavings as floor covering. The mice were maintained in a vivarium with controlled humidity and temperature (35–40%, 21 6 1°C) and a 12-h light/dark cycle (lights on at 8:00 AM). All procedures were conducted with the approval and under the supervision of the Tufts University Institutional Animal Care and Use Committee. The animals were cared for according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources,