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No effects of chlordiazepoxide on reactivity to conspecific or novel odors in mice.

Chlordiazepoxide (CDP) and related benzodiazepine compounds consistently reduced defensive or anxiety-like responses in wild and laboratory rodents over a wide range of testing situations (e. g., Blanchard, D. C., & Blanchard, R. J., 1990; Blanchard, D. C., Blanchard, R. J., & Rodgers, 1990; Blanchard, D. C., Hori, Rodgers, Hendrie, & Blanchard, R. J., 1989; File, 1990; Krsiak & Sulcova, 1990) and sometimes (e. g., Krsiak & Sulcova, 1990; Olivier, van Aken, Jaarsma, van Oorschot, Zethof, & Bradford, 1984; Panksepp, 1971), but not always (e. g., Krsiak, 1979; Miczek, 1974; Olivier & van Dalen, 1982; Rodgers & Waters, 1985) attenuated offensive attack. When found, reduced offense was often accompanied by increased social investigation (e. g., Krsiak, 1979; Krsiak & Sulcova, 1990; Olivier, 1981; Poole, 1973) during which sniffing was conspicuous. The latter observation suggested that altered responsiveness to conspecific odors may contribute to the antiaggressive effects of benzodiazepines (Dixon, 1982). Indeed, inhibition of attack by several drugs with differing pharmacological properties was accompanied by increased social sniffing and some disruption in normal olfactory function (Kemble, Behrens, Rawleigh, & Gibson, 1991; Kemble, Schultz, & Thornton, 1986; Olivier, 1981; Olivier et al., 1984; Ostrem, Rawleigh, & Kemble, 1992; Racine, Flannelly, & Blanchard, D. C., 1984; Soffie & Lamberty, 1988). The present experiments were therefore undertaken to explore the effects of CDP on reactivity to conspecific and novel odors. Odor preference or rejection was assessed with a three-choice preference test. In previous research, this task clearly revealed preferences for conspecific odors (Kemble & Gibson, 1992), which were reliably shifted by prior dominance or subordination experience (Rawleigh, Kemble, & Ostrem, in press), and a strong rejection of potential (cat) predator or novel odors (Kemble & Gibson, 1992).

General Method

Subjects

The subjects were experimentally naive male CD-1 albino mice. All subjects were individually housed on sawdust bedding and maintained on a 12-hr light/dark cycle. All testing was conducted during the light phase. Ad lib access to Purina Lab Chow and water was provided throughout the experiment.

Apparatus

Testing was conducted in a four-choice olfactory preference apparatus constructed of Plexiglas. Four 20- x 11.5- x 17.5-cm odor compartments extended from the center of each wall of a 30- x 30- x 17.5-cm central chamber. The most distal 10 cm of each odor compartment floor consisted of an aluminum plate having 84 equally spaced 0.4-mm perforations. A 9.3- x 9.7-cm Plexiglas tray containing odorous substances was placed beneath the aluminum floor of each odor compartment. Access to the odor compartments was provided by four independently controlled guillotine doors. Only three odors were used in these experiments, so one door remained closed during testing. A more detailed description of the apparatus is available elsewhere (Ostrem et al., 1992). The apparatus and all odor trays were thoroughly cleaned with detergent solution after each test. The positions of the odors were systematically varied over trials. Habituation and testing were conducted under dim red illumination and observations carried out from an adjacent room.

Odor Sources

Conspecific odors were provided by 50 ml of sawdust bedding which had been soiled for 21 days prior to testing. Each odor donor was housed on 800 ml of bedding/animal during this time. Unfamiliar male odors were provided by 16 singly housed male mice, and each dominant or subordinate subject donated odors for its familiar agonistic partner. Neutral odors were provided by 50 ml of unsoiled bedding. Novel odors were provided by 2.5 ml of either unsweetened powdered chocolate (Hershey) or cinnamon (Trader's Choice).

Procedure

Subjects were randomly assigned to weight-balanced groups designated to receive 2.0 mg/kg (Low) or 8.0 mg/kg (High) CDP or an equivalent volume of isotonic saline (Saline). The CDP dosages selected are well within the range known to inhibit defensive behavior effectively (e. g., Blanchard, D. C., et al., 1989; File, 1990; Olivier et al., 1984; Rodgers & Waters, 1985). The mice were habituated to the apparatus for 15 min/day for 2 days. On the day of testing, drugs or saline were administered by intraperitoneal injection 30 min prior to testing. Shortly (3 - 4 min) before testing, trays containing the odorous substances were placed beneath the perforated floors of the odor compartments. Subjects were placed in the central chamber and three guillotine doors opened simultaneously 5 - 10 sec later. The number of entries and time spent in each odor compartment were recorded for 10 min.

Responsiveness to each of the odors within groups were analyzed by a series of Wilcoxon signed rank tests and the drug groups compared by single factor analyses of variance. Number of entries were significantly increased at the high dose of CDP regardless of odor source in both experiments (ps < 0.05) suggesting drug-induced hyperactivity. Furthermore, within-groups frequencies were quite variable. Discussion will therefore be limited to duration data.

Experiment 1

Experiment 1 examined the effects of CDP on preferences for conspecific odors. Because prior agonistic experience is known to alter such preferences (e. g., Brown, 1992; Carr, Matarano, & Krames, 1970; Rawleigh et al., in press), we also wished to determine if drug effects might be differentially influenced by prior defeat or victory. Stable dominance/subordination relationships were therefore established by a series of resident-intruder encounters and preferences of dominants and subordinates for the odors of their agonistic partner and for unfamiliar conspecifics were compared to those for unsoiled sawdust.

Method

Twenty-eight pairs of male mice (Low CDP = 9, High CDP = 10. Saline = 9) which had been individually housed for 21 days served as subjects. One member of each pair was designated as the resident (alpha) male and weighed 4.0 - 5.0 g more than its subordinate partner. At the end of social isolation, three daily agonistic encounters (Day 1 = 10 min, Days 2 and 3 = 5 min) were staged by placing the subordinate member of each pair into the home cage of the resident alpha. Residents and intruders were tested with the same agonistic partner on all three encounters. All residents displayed species-typical attack to which subordinates responded defensively. Responses to the soiled bedding odors of familiar antagonists, unfamiliar males, and unsoiled sawdust were compared 48 hr after the final agonistic encounter.

Results

All subordinate groups showed preferences for alpha (Ms = 173 - 186 sec) and unfamiliar conspecific (Ms = 142 - 153 sec) odors when compared to sawdust (Ms = 89 - 120 sec, ps < .10 - .01). Both CDP-treated alpha groups also preferred subordinate (Ms = 152 - 177 sec, ps < .05 - .01) and unfamiliar conspecific (Ms = 131 - 164 sec, ps < .10 - .05) odors to sawdust (Ms = 86 - 120 sec). Although saline-treated alphas also spent more time in subordinate (M = 152 sec) and unfamiliar conspecific (M = 151 sec) compartments than sawdust (M = 120 sec), these differences were not statistically significant (p > .10). Group comparisons failed to reveal any effects of drug treatment on responsiveness to any of the odors (ps > . 10).

Experiment 2

Although CDP was without effect in Experiment 1, it should be noted that neither conspecific odor was avoided. Because CDP quite consistently reduces defensiveness; (e. g., Blanchard, D. C., & Blanchard, R. J., 1990; Blanchard, D. C., et al., 1990; Olivier et al., 1984), it seemed possible that drug effects might be restricted to aversive odors. Experiment 2 examined the effects of CDP on reactions to two novel odors which were previously found to be strongly rejected in this task (Kemble & Gibson, 1992).

Method

Thirty subjects weighing 28.0 - 41.4 g were individually housed in 25.0-x 19.0- x 13.0-cm stainless steel cages having a sawdust substrate for 7 days prior to testing. The subjects were randomly designated to receive low CDP (N = 11), high CDP (N = 9), or saline (N = 10). Following habituation to the apparatus, reactivity to the odors of chocolate, cinnamon, and unsoiled sawdust were measured as previously described.

Results

All groups showed a significant rejection of cinnamon odor (Ms = 78 - 88 sec) when compared to sawdust (Ms = 147 - 168 sec, ps < .05 - .01). Chocolate odors were rejected only at the highest CDP dosage, however (M = 93 sec, p < .05). Group comparisons did not reveal any reliable drug effects on responsiveness to the novel odors (ps > .10).

Discussion

The present experiments failed to reveal any effect of CDP on responsiveness to conspecific or novel odors. These findings stand in apparent contrast to those of Blanchard, R. J., Blanchard, D. C., Weiss, and Meyer (1989) who found that diazepam-treated rats markedly reduced "risk assessment" behaviors (i. e., flat back approaches, stretch attends) in response to cat odors. Of course, the use of CDP rather than diazepam might account for these differences. It might also be argued that the failure of CDP to alter reactivity in Experiment 2 resulted from the use of highly artificial odors having no ethological relevance to the subjects. In earlier preference tests, however, we (Kemble & Gibson, 1992) found that mice showed a strong and equivalent rejection of soiled cat litter, unsoiled cat litter, chocolate, and cinnamon odors. Brief exposure to unsoiled cat litter or cinnamon odors also increased shock thresholds (Kemble & Gibson, 1992) and chocolate odor increased the incidence of fleeing by residents during a resident-intruder encounter (Kemble & Garbe, 1993). Taken together, these data clearly indicate that novel odors are effective fear-inducing stimuli. Furthermore, if CDP effects are restricted to ethologically relevant stimuli, it is difficult to see why preferences for conspecific dominance and/or subordination odors, which are elevated by prior stress or defeat (Williams & Groux, 1993), were not affected in Experiment 1.

Alternatively, the apparent discrepancies between the present findings and those of Blanchard, R. J., et al., (1989) may reside in either the nature of the behaviors recorded or the behavioral task. It is possible that CDP did, in fact, alter risk assessment but that such behavioral changes did not alter duration of contact with the conspecific and/or novel odors. Indeed, both flat back and stretch attend responses were noted during these experiments. Observers were unaware of odor positions and drug conditions (to preclude any observer bias), therefore it is not possible to determine if the drug had selective effects on these behaviors. It is also possible that the test itself obscured drug effects. In subsequent research we (Garbe, Kemble, & Rawleigh, in press) found that mice showed very low levels of risk assessment to novel odors when testing was conducted in a straight runway with the odor source located at one end. When testing was conducted in the animals' home aquaria with the odor stimuli scattered over half of the sawdust floor, however, novel odors induced a dramatic increase in risk assessment. Because the preference apparatus used in these experiments was similar in some ways to a runway apparatus, it may be that this task is ill-suited to reveal odor-induced fear in mice. Direct observation of species--typical fear responses to such odors in a familiar environment would be useful in future studies of drug effects. In any case, the fact that several other antiaggressive drugs effectively alter odor preferences in similar tests (Kemble et al., 1986, 1991; Ostrem et al., 1992; Soffie & Lamberty, 1988) suggests that CDP's anxiolytic effects may not be mediated by major shifts in the attractiveness or aversiveness of odors.

Please address correspondence to Ernest Kemble, Division of Social Sciences, University of Minnesota-Morris, Morris, MN 56267. This research was partially supported by a sabbatical leave to Ernest D. Kemble.

References

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Author:Garbe, Colleen M.; Rawleigh, Joyce M.; Kemble, Ernest D.; Bursell, Amy L.
Publication:The Psychological Record
Date:Mar 22, 1993
Words:2574
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