Psychopharmacology (2002) 163:488–494 DOI 10.1007/s00213-002-1135-x

O R I G I N A L I N V E S T I G AT I O N

Jeffrey M. Witkin

Some contextual and historical determinants of the effects of chlordiazepoxide on punished responding of rats

Received: 3 November 2001 / Accepted: 24 April 2002 / Published online: 7 June 2002 © Springer-Verlag 2002

Abstract Rationale: A host of factors that modulate the increases produced by benzodiazepines on responding suppressed by punishment have been described. Nonetheless, the necessary and sufficient conditions for the anxiolytic-like activity in this animal model have not been fully delineated. Objectives: The present experiments sought to determine the necessity of the reinforcing event (food delivery), the role of the relationship of food delivery to the punishing stimulus, and the prevailing historical context of behavior in determining the effects of chlordiazepoxide (CDAP) from 1 to 17 mg/kg, i.p. on punished responding. Methods: Male, Sprague-Dawley rats pressed a lever under a multiple schedule. In the presence of one stimulus, every 30th response produced food and, in the presence of an alternate stimulus, every 10th response produced food, a brief electric shock, or food plus shock. Additionally, the baseline schedule was manipulated to determine antecedent experience that may contribute to the efficacy of CDAP. Results: Chlordiazepoxide generally produced little or no effect under the FR 30 schedule but increased response rates under the FR 10 schedule when responding produced either food plus shock (to 600% of control) or shock alone (300% of control) but not food alone. The increases produced when shock alone was delivered were eliminated when rats did not have a history of food plus shock pairings. In addition to increasing suppressed responding, CDAP also prevented the suppression in both punished and non-punished response rates that resulted from adding a food plus shock or shock alone contingency. Conclusions: Chlordiazepoxide and perhaps benzodiazepines in general have robust efficacy for both reducing response suppression and for preventing its occurrence. This efficacy is modulated by conditions present at the time of drug exposure and by the history of the organism with respect to response contingencies. J.M. Witkin (✉) Neuroscience Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285-0510, USA e-mail: [email protected] Tel.: +1-317-2774470, Fax: +1-317-2767600

Keywords Chlordiazepoxide · Punishment · Anxiolytic drug · Rat

Introduction Conceptualizations of punishment have historically focused on motivational and emotional constructs. However, an analysis of behavior in behavioral terms as initially advocated by Peter Dews (Dews 1958) is essential for orderly relationships to emerge between behavior and drug effects (cf. Kelleher and Morse 1968). Punished behavior has thus been defined as “a reduction in the future probability of a specific response as a result of the immediate delivery of a stimulus for that response” (Azrin and Holz 1966). Responding suppressed by punishment has received much scrutiny by behavioral pharmacologists partly due to the impressive specificity of drug action under punishment baselines, the observation that amphetamines, well known for increasing low rates of responding (Dews and Wenger 1977), do not generally increase the low rates of responding suppressed by punishment (Geller and Seifter 1960; see also McMillan 1975 for a summary), and because increases in punished responding are predictive of clinical efficacy for the management of anxiety (Geller et al. 1962; Geller 1964). Thus, the potencies of drugs to increase punished responding correlates positively with average clinical dose (Cook and Davidson 1973). In addition to neuropharmacological and a neuroanatomical information (cf. Gray 1982; Sanger 1994), a host of environmental and historical determinants of the effects of drugs on punished responding have also been revealed (cf. Kelleher and Morse 1964, 1968; McMillan 1975). These variables not only can determine whether a drug produces an effect or not but can determine the overall specificity of the drug with respect to non-punished behavior. For example, methaqualone has been shown to either increase punished responding more, less, or equivalently to equal rates of non-punished responding depending upon the intensity of the punishing stimulus (observa-

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tions of J.M. Witkin, J.L. Katz, and J.E. Barrett cited in Witkin and Katz 1990). As Kelleher and Morse (1968) pointed out, the specificity of drug effects on behavior is the critical aspect of their profile that is of interest to behavioral and clinical psychopharmacologists. As such they emphasized that elucidation of the determinants of such specificity should be the principle effort of behavioral pharmacology. The goal of the present experiment was to evaluate some of the necessary and sufficient conditions for the increases in suppressed responding by chlordiazepoxide (CDAP). Lever pressing of rats was maintained under various multiple schedules. In the presence of one stimulus, every 30th response produced food and, in the presence of an alternate stimulus, every 10th response produced a consequence. The event(s) delivered contingent upon completion of the FR 10 schedule were varied [mult FR 30(f) FR 10(x) schedule]. Additionally, the baseline schedule was manipulated to determine antecedent experience that may contribute to the efficacy of CDAP.

Materials and methods Animals Male, Sprague-Dawley rats (Zivic Miller Laboratories, Allison Park, Pa., USA) were maintained at 350±18 g by postsession feeding. Food and water were continuously available for all animals in their individual living cages that were housed in a temperaturecontrolled room. Experiments were conducted during the light phase of a 12-h light/dark cycle. The facilities in which the animals were maintained are fully accredited by the American Association for the Accreditation of Laboratory Animal Care, and the studies described herein were conducted in accordance with the Guide for Care and Use of Laboratory Animals. Apparatus Experiments were conducted in standard two-lever rat experimental chambers (Coulbourn Instruments, Leigh Valley, Pa., USA). Depression of the right lever with a force exceeding 35 g (0.35 N) defined a response and produced the audible click of a relay. The chambers were located within sound-attenuating cubicles supplied with ventilation and white noise to mask extraneous sounds. Scrambled electric shock could be delivered to the grid floor of the chamber by a constant current AC source. Experimental events and data were collected with a PDP 11/73 computer operating under SKED software (State Systems, Kalamazoo, Mich., USA). Behavioral procedure Groups of six rats each were trained under various baseline conditions of reinforcement. When responding was stable, effects of CDAP were determined either under the same conditions as in training or under conditions different from baseline training. The specific experimental manipulations made during drug testing are described in detail in Results. All rats were initially trained under a multiple fixed-ratio, fixed-ratio schedule in which every 30th lever press in the presence of green lever lights produced food and every 10th lever press in the presence of red lever lights produced food (45 mg pellet; BioServe, Frenchtown, N.J.,USA) as described in detail (Witkin and Perez 1990). The fixed-ratio requirement was counted from the onset of each schedule component that lasted for 3 min. Schedule components were separated by 30-s timeout periods; during timeout, all lights were extinguished and responding had no scheduled consequences. Experimental ses-

sions began with the green light and lasted for five alternate presentations of components signaled by green (FR 30) and red (FR 10) lights. After training, the baseline conditions were changed for some groups such that a schedule of electric shock delivery (200 ms) was put in effect under the FR 10 schedule component conjointly with food delivery. The shock intensities were adjusted for each rat (0.5–1.75 mA) to suppress responding to about 10% of non-punished response levels. Each single, fixed duration experimental session was conducted daily, 5 days per week. All doses of CDAP were studied in all rats; a range of doses of CDAP (1–17 mg/kg) or vehicle was generally studied once in each rat in a mixed order. Injections were given on Tuesday or Friday provided that responding was stable on the day prior to injection. Data from sessions conducted on Thursday were used as non-injection control sessions for data evaluation. Thus, the baseline schedule was in effect on days when CDAP was not given. On experimental sessions in which CDAP was studied, the conditions during a schedule component or components either remained in effect (experiments 1 and 5) or were changed (experiments 2–4, 6, and 7). Each condition was studied until full dose-response curves were generated. Data analysis Rates of responding under each component of the multiple schedule in the absence of drug were averaged individually for each rat. Drug effects for each rat were expressed as a percentage of their individual baseline, non-injection values. These percentages were then averaged across rats to obtain composite doseeffect curves for a group of six rats. The mean data for the group was evaluated with ANOVA followed by post hoc Dunnett’s test using vehicle as the control standard. Statistical probabilities of less than 0.05 were considered to be significant. In addition, drug effects in each individual rat were analyzed. Drug effects for individuals were considered significant when they deviated by at least 2 SD of rates engendered when saline was given. Drugs Chlordiazepoxide HCl (Hoffmann-LaRoche, Nutley, N.J., USA), mixed freshly each day, was dissolved in 0.9% NaCl and injected (i.p.) in a volume of 1 ml/kg, 30 min prior to behavioral testing. Drug doses are expressed as the salt.

Results Punished responding An experimentally naïve group of rats was trained under a multiple schedule of non-punished and punished responding. Responding under the mult FR 30(f) FR 10(f+s) schedule engendered high rates of responding in the component in which responding produced food and low rates of responding in the component in which responses produced both food and shock, FR 30=2.5±0.16 responses/s and FR 10=0.16±0.03 responses/s (mean ± SEM). Chlordiazepoxide produced dose-dependent increases in punished responding without affecting non-punished response rates up to 17 mg/kg (Fig. 1). All rats exhibited significant increases after at least one dose of CDAP. Elimination of food delivery This experiment evaluated the role of food delivery during the punishment component as a variable controlling

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Fig. 1 Effects of chlordiazepoxide HCl (CDAP) on rates of nonpunished [FR 30(food)] and punished responding [FR 10(food + shock)]. The baseline schedule prior to drug testing was the same as when CDAP was administered mult FR 30(food) FR 10(food + shock). Unfilled bars Responding under the FR 30 schedule, filled bars responding under the FR 10 schedule. Each bar represents the mean ± SEM of six rats expressed as a percentage of baseline noninjection control values. Significant differences (P<0.05, Dunnett’s test) from vehicle control values (bars above 0) are represented by an asterisk

Fig. 3 Effects of CDAP on rates of non-punished [FR 30(food)] and suppressed responding [FR 10(shock)]. The baseline schedule prior to drug testing was the same as when CDAP was given: mult FR 30(food) FR 10(shock). Other details as in Fig. 1

rats displayed significant increases after at least one dose of CDAP. The maximal increases produced by CDAP under these conditions, however, were about one half that achieved when responding in the presence of CDAP produced both food and shock (compare vehicle to drug effect in Fig. 1 versus Fig. 2). Non-punished responding under the FR 30 component was also unaltered up to 10 mg/kg. Elimination of a history of food + shock pairings

Fig. 2 Effects of CDAP on rates of non-punished [FR 30(f)] and suppressed responding [FR 10(shock)]. The baseline schedule prior to drug testing was a mult FR 30(food) FR 10(food + shock). Other details as in Fig. 1

the increases in rates of responding in that component. For this purpose, the rats trained under the mult FR 30(f) FR 10(f+s) schedule above were studied with CDAP as in the previous experiment, however, in the present experiment, shock but not food was presented under the FR 10 schedule only when drug or vehicle was administered. Under these conditions, rates of responding under vehicle were not significantly altered; high rates of responding continued to be maintained under the FR 30(f) component and low rates of responding occurred under the FR 10(s) component (FR 30=3.3±0.21 responses/s; FR 10=0.11±0.06 responses/s). As under the standard punishment baseline, CDAP also increased response rates when responding only produced shock (Fig. 2). All

In the previous experiment, CDAP increased suppressed responding even when the consequence of rate increases was to increase the rate of shock delivery in the absence of increases in food presentations. In order to determine if the baseline of food plus shock pairings under the punishment baseline was responsible for the increases observed with CDAP, a separate group of rats was trained under a mult FR 30(f) FR 10(s) schedule from the outset. They were maintained under these conditions when CDAP was administered. Baseline rates of responding under this baseline were FR 30=2.9±0.18 responses/s and FR 10=0.09±0.02 responses/s. Chlordiazepoxide did not significantly increase responding under these conditions and indeed produced decreases in responding at doses of both 10 and 17 mg/kg (Fig. 3). Although significant increases were not observed at any dose of CDAP, individual rats did exhibit increases in responding that resulted only in shock delivery. Increases occurred in three rats at 3 mg/kg, and two rats at both 5.6 and 10 mg/kg. To explore the influence of the non-punished response component on these increases, a dose-effect curve for CDAP was obtained when the nonpunishment component was eliminated in a separate group of rats. These rats were first trained under a mult FR 30(f) FR 10(f) schedule, subsequently under a mult FR 30(f) FR 10(f+s), and finally under a mult FR 30(f)

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Fig. 4 Effects of CDAP on rates of responding under an FR 10(shock) schedule without an alternate schedule component. Prior to CDAP administration, responding had been maintained under a mult FR 30(food) FR 10(shock) schedule. Other details as in Fig. 1

Fig. 6 Effects of CDAP on rates of non-punished [FR 30(food)] and suppressed responding [FR 10(food + shock)]. The baseline schedule prior to drug testing was a mult FR 30(food) FR 10(food) schedule. Other details as in Fig. 1

Does CDAP prevent punishment?

Fig. 5 Effects of CDAP on rates of non-punished responding under a mult FR 30(food) FR 10(food) schedule. The baseline schedule prior to drug testing was the same as under drug testing. Other details as in Fig. 1

FR 10(s) schedule baseline as described in Fig. 3. Under the FR 10(s) component alone when presented alone, CDAP only decreased rates of responding in all rats (Fig. 4).

Using the multiple schedule without punishment above [mult FR 30(f) FR 10(f)] as a baseline, CDAP was administered under conditions in which electric shock was introduced into the FR 10 component [FR 10(f+s)]. Under the baseline schedule of food delivery, rates under the FR 30(f) were 2.6±0.11 responses/s and under the FR 10(f) schedule, rates were 2.6±0.13 responses/s. Introduction of the punishment contingency produced marked decreases in rates of responding under both components of the multiple schedule with the largest effect seen under the punishment component (0 mg/kg in Fig. 6). Chlordiazepoxide significantly attenuated the response suppression induced by addition of the shock contingency. Protection was seen for both the punishment and non-punishment components. All rats displayed significant increases after at least one dose of CDAP. Note, however, that although rates in the punishment component were substantially increased above vehicle control levels by doses of 5.6–17 mg/kg chlordiazepoxide, responding never increased to the rate occurring when no shock was present (100% in Fig. 6). Elimination of the food contingency

No punishment The necessity for responding to be suppressed or occurring at a low rate was investigated under conditions without punishment. A separate group of rats without prior experience was trained and maintained under a mult FR 30(f) FR 10(f) schedule. Under this baseline, rates of responding under both components were high: FR 30=2.5±0.12 responses/s and FR 10=2.7±0.14 responses/s. Chlordiazepoxide only decreased the high rates of responding engendered under both the FR 30 and FR 10 schedule components of food delivery (Fig. 5).

Chlordiazepoxide was able to protect against the response suppression engendered by addition of the punishment contingency as noted in the experiment immediately above. The present experiment addressed whether CDAP was also capable of preventing the response suppression induced by the presentation of shock alone in the absence of food delivery. A separate group of rats was maintained under a mult FR 30(f) FR 10(f) and probed with doses of CDAP when shock replaced food as the consequence for responding under the FR 10 schedule component [mult FR 30(f), FR 10(s)]. Prior to exposure to shock, rates under the FR 30(f) schedule

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Fig. 7 Effects of CDAP on rates of non-punished [FR 30(food)] and suppressed responding [FR 10(shock)]. The baseline schedule prior to drug testing was a mult FR 30(food) FR 10(food) schedule. Other details as in Fig. 1

were 2.8±0.9 responses/s; FR 10=2.9±0.10 responses/s. As with the introduction of the food plus shock contingency above (Fig. 6), replacement of food with electric shock resulted in large decreases in rates of responding under both schedule components (vehicle or 0 value, Fig. 7). When CDAP was given in conjunction with the change in contingency, rates of responding were decreased less; thus CDAP protected against the suppressant effects of adding a response produced shock contingency. All rats displayed significant increases after at least one dose of CDAP.

Discussion The benzodiazepine anxiolytic, CDAP, increased responding suppressed by punishment, a finding consistent with many prior investigations (cf. Geller et al. 1962; Barrett et al. 1985; Witkin and Perez 1990; see also Cook and Davidson 1973 for review). In the present series of experiments, some of the conditions required for this effect were evaluated by selective exclusion of events during drug testing and by manipulation of experiential history. It was found that robust increases in suppressed responding could be produced not only when responding produced concomitant food and shock presentation but also when responses produced shock as the only consequence. In order for CDAP to increase responding when shock was the sole consequence, a history of food plus shock pairings were necessary. However, even in the absence of such pairings, there were individual rats that exhibited increases after CDAP with just a history of response-produced shock presentation. The increases seen in individual rats after a shock only history were dependent upon the context of the multiple schedule under which responding in an alternate component produced food. This result differs from the continued maintenance of drug-induced increases in punished responding in the absence of an alternate no punishment component (Kelleher and Morse 1964).

In the present experiment, qualitative changes in the effects of a drug were made by alterations in the behavioral experience prior to drug exposure. That behavioral history and behavioral context were capable of modulating the effects of CDAP on punished responding was not surprising as other examples of historical modulation of drug effects on punished responding have been observed (cf. Barrett and Witkin 1986). For example, although d-amphetamine does not generally increase punished responding, increases can be observed in animals responding within specific behavioral contexts (McKearney and Barrett 1975) or after specific histories (Barrett 1977; Barrett and Witkin 1986). In the current study, CDAP increased responding that produced electric shock alone after a history of food plus shock pairings but not after a history of response-produced shock alone. The CDAP-induced increases in responding that produced shock alone occurred in rats that had previously been given CDAP under conditions of response-produced food plus shock. Since CDAP increased rates of responding, rates of food delivery were also increased. It is thus possible that the prior history with CDAPinduced increases in food presentations played a role in driving rate increases when shock alone was the response consequence. Chlordiazepoxide previously studied under the FR 10(f) schedule did not increase rates of food delivery. In these animals, CDAP still increased rates of responding under the FR 10(s) schedule. Thus, a history of CDAP-induced increases in food delivery is not necessary for the increases observed under the FR schedule of response-produced shock. Nonetheless, it is not known whether the increases produced by CDAP when shock alone is presented would be sustained over repeated drug exposure in the absence of retraining with food. Although this is the first report to show that a benzodiazepine can increase suppressed responding when the only consequence was electric shock delivery, previous studies have demonstrated that food delivery is not necessary for CDAP to increase the low rates of responding engendered under other conditions. For example, in the context of a multiple schedule of punished and non-punished responding, responding without scheduled consequences (extinction) occurs at a low rate and can be increased by CDAP (Hanson et al. 1967; Moore et al. 1994) although this effect is not always significant (Miczek 1973). Deletion of food delivery during CDAP testing resulted in a diminished response to the drug in the present experiment; nonetheless, large and significant increases were still observed. Although the precise magnitude of difference cannot be addressed here, the general difference in drug efficacy is significant as CDAP and related compounds continue to produce large increases in responding even with daily administration as previously reported (Margules and Stein 1968; Witkin and Barrett 1981). Finally, although food delivery per se may not be necessary for the increases in suppressed responding by CDAP [FR 10(s)], the environmental context of food delivery is necessary for CDAP to produce

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increases [for example, no increases under FR 10(s) but increases under mult FR 30(f) FR 10(s)] even when control rates of suppressed behavior are held largely constant. In addition to increasing suppressed responding, CDAP also diminished the response suppression produced by introduction of either the food plus shock contingency or the shock alone contingency. This dampening effect on the suppressive effects of punishment is similar to that occurring under the Vogel conflict test using water drinking as the operant response (Vogel et al. 1971). It remains to be seen whether the use of food presentation generates false positives as with isoproterenol in the Vogel procedure with water reinforcement (Patel and Malick 1980). These prophylactic data are also consistent with the literature showing attenuation of stressinduced changes in behavior and stress hormones by benzodiazepines (Vogel et al. 1984; Kitaichi et al. 1995; Zethof et al. 1995; Sanna et al. 1999). These findings are also in accord with the clinical efficacy of benzodiazepines in reducing the impact of anxiety-provoking stimuli on stress hormones, immunological responses to stress and other potential health markers and behavior (Williams 1990; Benschop et al. 1996; Zavala 1997). Indeed, the historical and contextual modulation of the anxiolytic-like effects of CDAP may be based upon compensatory changes in GABA receptor function. For example, neuroactive steroids are altered by stressors such as electric shock and, through their interaction with their own receptor on the GABAA receptor complex, produce anxiolytic effects in a number of distinct of animal models (see Gasior et al. 1999). A host of stressors are also well known to regulate GABAA receptor sensitivity through direct modulation of receptor densities (cf. Hommer et al. 1987), by modulation of glucocorticoid function (cf. Cullinan and Wolfe 2000), and likely via a host of other mechanisms relating to GABA (see, for example, Kash et al. 1999) and non-GABA receptormediated processes (for example, CRF, glutamate, NPY, etc.). The findings of the present study add to the literature on the necessary and sufficient conditions for CDAP, and perhaps positive allosteric modulators of GABA in general, to increase responding suppressed by punishment. Both current and past environmental conditions influence the behavioral effects of this anxiolytic agent. As Kelleher and Morse pointed out over 30 years ago, "...these procedural differences are more important determinants of the actions of a drug than the supposed common component of anxiety” (Kelleher and Morse 1968 p 40). It is these procedural differences (prevailing environment, context, behavioral history, etc.) that must be scrutinized as they undoubtedly have a critical bearing upon clinical response. Acknowledgements The author is grateful for the pioneering work and writings of Peter B. Dews, William H. Morse, and Roger T. Kelleher and for their personal influence on my behavior and their indirect influence through the mentoring by those they have influenced.

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