The testosterone metabolite and neurosteroid 3alpha-androstanediol may mediate the effects of testosterone on conditioned place preference.

The abuse of androgens may be related to their ability to produce positive, hedonic interoceptive effects. Conditioned Place Preference (CPP) has been used in many experiments to examine hedonic effects of drugs. This review is focused on studies from our laboratory that utilized CPP to examine potential positive hedonic effects of testosterone (T), and its androgenic metabolite dihydrotestosterone (DHT), and its metabolite 3alpha-androstanediol (3alpha-diol). We hypothesized that administration of a high concentration of 3alpha-diol would produce a CPP, pharmacological concentrations of plasma androgens, and alter androgen receptors (AR) and the function of GABA(A)/benzodiazepine receptor complexes (GBR). In our studies, we observed that systemic 3alpha-diol (1.0 mg/kg) prior to exposure to the non-preferred side of a CPP chamber significantly increased preference for the non-preferred side of the chamber compared to baseline preference and homecage controls. Furthermore, administration of T, DHT, or 3alpha-diol increased levels of these androgens, decreased ARs (decreased seminal vesicle weight and intrahypothalamic AR) and GBR function (decreased GABA-stimulated chloride influx in cortical synaptoneurosomes, and muscimol binding in the hippocampus compared to control groups). With systemic administration of 3alpha-diol that enhanced CPP, concentrations of 3alpha-diol were increased in the nucleus accumbens (NA). Central implants of T, DHT, or 3alpha-diol to the NA also produced a CPP compared to baseline preference and vehicle controls. These data indicate that systemic 3alpha-diol is more effective at enhancing CPP and increasing circulating 3alpha-diol levels than is T or DHT and that central administration of 3alpha-diol to the NA can condition a place preference. These findings indicate that 3alpha-diol produces positive hedonic effects and suggest that T's variable effects on CPP may be due in part to T's metabolism to 3alpha-diol.

Sex differences in relation to conditioned fear-induced enhancement of morphine analgesia.

A number of studies have reported that both the immediate and proactive effects of exposure to a shock stressor are less pronounced in female than in male rats. A separate area of research has demonstrated that female rats are less sensitive to the analgesic effects of morphine than males. Experiments from our laboratory, as well as others, have found that exposure to a context associated with shock (i.e., conditioned fear context) at the time of morphine administration, enhances the analgesic effects of morphine. Since previous studies have exclusively employed male rats, the purpose of Experiment 1 was to determine if a sex difference exists to this context conditioned fear-induced enhancement of morphine-induced analgesia. The findings of Experiment 1 showed that females do not appear to exhibit conditioned fear-induced enhancement of morphine analgesia as compared to males. Experiment 2 demonstrated that females exhibited higher levels of conditioned fear-induced enhancement of morphine analgesia during diestrus I than estrous. Experiment 3 demonstrated that females exhibited lower levels of conditioned analgesia compared to males, while both groups exhibited similar freezing levels. The findings of the present experiments suggest that the sex difference observed in Experiment 1 may be due to differences in conditioned analgesia.

Pavlovian aversive context conditioning using carbon dioxide as the unconditional stimulus.

Four experiments were conducted to examine the utility of carbon dioxide (CO2) as an aversive unconditioned stimulus (US) in a Pavlovian context conditioning paradigm. Experiment 1 demonstrated that rats exposed to CO2 in a distinctive context showed elevated levels of freezing relative to controls. Experiment 2 replicated this basic effect with a modified conditioning procedure and additionally demonstrated conditioned analgesia. Experiment 3 demonstrated a positive monotonic relationship between US duration and resistance to extinction of freezing behavior as well as conditioned analgesia. Experiment 4 demonstrated extinction and an extinction-related phenomenon, renewal. These studies clearly demonstrate the utility of CO2 as a Pavlovian US.

Methotrexate does not interfere with an appetitive Pavlovian conditioning task in Sprague-Dawley rats.

There has been considerable interest in developing an animal model of the neuropsychological toxicity of chemotherapeutic agents used in the treatment of patients with cancer, especially children, since these agents often cause significant, long-term neuropsychological deficits. Yanovski, Packer, Levine, Davidson, Micalizzi, D'Angio (13) recently proposed such a model based on their finding that methotrexate retarded the formation of aversive Pavlovian excitatory associations. The present experiment examined the generality of methotrexate induced cognitive impairments by testing rats in Appetitive Pavlovian Conditioning tasks and a Conditioned Taste Aversion paradigm. The results of our study revealed no impairment following methotrexate exposure on the Appetitive Pavlovian tasks or on the Taste Aversion task, relative to two control conditions. While there were a number of methodological differences between the present experiment and those conducted by Yanovski et al. (13), the present results question the robustness and generality of Yanovski's et al. (13) animal model.

Conditioned fear exacerbates acute morphine dependence.

A variety of physical stressors have been shown to enhance reactivity to opioid drugs. Few studies have examined the effects of nonphysical stressors on opioid drug reactivity. In this regard, it has previously been shown that animals administered morphine in the presence of shock-associated cues demonstrate increases in hypoalgesia relative to nonshock control animals. These findings have typically been viewed as being mediated by the activation of endogenous pain inhibition systems via conditioned fear. In this series, we further examined the nature of these effects by assessing the effects of conditioned fear on acute morphine dependence. Experiment 1 revealed that animals administered 3 mg/kg morphine in the presence of context fear cues demonstrated an enhanced withdrawal response when removed and administered 3 mg/kg naloxone. Because it is known that conditioning effects do not diminish over time, a second experiment examined whether the enhancement of acute dependence by context fear would still be evident 72 h postconditioning. As in Experiment 1, animals administered morphine in a context associated with shock demonstrated an enhancement of acute dependence. Experiment 2b revealed that the shock parameters used in these studies can induce a hypoalgesic response on the test that is opioid mediated. These findings are discussed with regard to the neuroanatomy of fear systems as they relate to the neuropharmacological study of opioid withdrawal.

Modulation of hypoalgesia by morphine and number of shock trials: covariation of a measure of context fear and hypoalgesia.

In a recent series of studies, we observed that exposure to prolonged foot shock increased hypoalgesia induced by morphine. This increase was observed only when testing was conducted in the presence of shock-associated cues, suggesting that it resulted from context-conditioned fear. However, we do not know whether the extended stressor parameters employed in that study are necessary for an observance of the effect. Therefore, in the present study, we assessed the effect of the number of shock trials (either 0, 20, 100, or 200) on the hypoalgesia observed following morphine administration. In addition, we measured activity as an independent index of context-conditioned fear, because in prior studies there had been no independent behavioral assessment of the conditioning of fear to the context. Although others have shown a covariation of conditioned fear and context-induced hypoalgesia using shock parameters and test paradigms different from our own, we sought to assess whether the same covariation would hold for conditioned fear and the hypoalgesia observed following the administration of morphine. The results showed increased hypoalgesia in all groups exposed to foot shock, demonstrating that prolonged exposure to foot shock is not necessary for an observance of this effect. In addition, the results revealed a linear relationship between number of trials of shock and hypoalgesia, but a U-shaped relationship between trials and activity. The pattern of results is considered in light of Fanselow's Perceptual-Defensive-Recuperative model.

Learned helplessness inducing foot shock can exacerbate morphine responsiveness.

Exposure to inescapable tail shock or foot shock has been shown to produce effects on a number of learning tasks. Tail-shock exposure is also known to influence nociception and morphine reactivity. The present series of experiments investigated the effects of foot shock known to induce learned helplessness effects in our laboratory on the subsequent reactivity to morphine. A first set of experiments investigated the hypoalgesic response to a 4 mg/kg dose morphine over 4 consecutive days following exposure to foot shock. Experiment 1A did not reveal an effect of foot shock on morphine-induced hypoalgesia when testing was conducted in a novel context. In Experiment 1B, we observed an increased hypoalgesic response to morphine when testing was conducted in the shock context. The findings of Experiment 1B were replicated in Experiment 2 and extended to assess the contribution of conditioned fear hypoalgesia to these effects. The possible mechanisms responsible for these findings are discussed.

Investigation of anabolic steroids in two taste aversion paradigms.

The aversive effects of estradiol have been studied in two different taste aversion paradigms. A similar investigation was undertaken for the anabolic-androgenic steroids, nandralone and testosterone cypionate, using Rockland-Swiss mice. Experiments 1 and 2 used the brief exposure of a novel saccharin solution as the conditioned stimulus for taste aversion learning, and showed that anabolic steroids (1 mg) do not induce taste aversions. Instead, these hormones induced a small non-contingent increase in saccharin preference. Experiment 3 showed that daily nandralone administration (1 mg/day) had a greater anabolic effect than the same dose of testosterone cypionate. Experiment 4 paired the continuous exposure to a novel diet with daily nandralone injections, and showed that steroid treatment increased intake of the novel diet. When the novel diet was subsequently presented with the familiar diet in a two-choice preference test, there was no indication that an aversion was conditioned to the novel target diet. On the contrary, nandralone treatment significantly increased the preference for the novel diet. These experiments show that anabolic-androgenic steroids do not have aversive effects in mice, and that they may have positive consequences.

Environmental enrichment: the influences of restricted daily exposure and subsequent exposure to uncontrollable stress.

Environmental enrichment has been proposed to enhance an animal's subsequent ability to learn. While this proposal has received considerable support from experiments involving maze tasks, it has received equivocal support from experiments employing operant and pavlovian tasks. The purpose of the present study is two-fold. The first is to demonstrate that a regimen of restricted daily exposure to environmental enrichment is capable of producing effects similar to those using more standard exposure regimens when compared to the most appropriate control, a group given social exposure. The second is to examine the proposed learning enhancement of environmental enrichment on an operant task both before and following exposure to uncontrollable stress. Uncontrollable stress, as interpreted by learned-helplessness theory, results in the formation of an expectancy of response-reinforcer independence which proactively interferes with the subsequent acquisition of response-outcome associations. It may be possible, then, that environmental enrichment and uncontrollable stress may interact in such a way as to allow the potential learning effects of environmental enrichment to be assessed on an operant task. Rats were exposed to differential environments; one group exposed to an enriched environment and another exposed to a social environment 2 hours daily for 30 days. Each group was then tested on the object-exploration test. Following the acquisition of an appetitive-operant response, a subset of these two groups was exposed to either controllable, uncontrollable, or no stress using parameters known to induce learned helplessness. Animals were then tested on an appetitive-noncontingent test. It was found that, while the enrichment procedure was effective in producing effects on the object-exploration test, environmental enrichment did not modify the acquisition of the operant or the effect produced by uncontrollable stress on the appetitive-noncontingent test.

Controllability of stressors and rewarding brain stimulation: effect on the rate-intensity function.

Previous research in mice has shown that an uncontrollable stressor, but not a controllable stressor, depresses responding for rewarding electrical stimulation of the brain (ESB) (33). We reexamined this effect in rats by determining rate-intensity functions for rewarding brain stimulation 24 h and 48 h following stressor exposure. In each rat, two electrodes, one on each side of the brain, were aimed at the posterior, lateral hypothalamus. Self-stimulation rate-intensity functions were obtained for each electrode; stimulation of one electrode always caused stronger activity than equivalent stimulation of the other. If the rats had been stressed with uncontrollable shock the preceding day, their responding was significantly depressed to stimulation at the weaker electrode site but not the stronger electrode site. However, if the rat had control over the stressor, then rate-intensity functions were unchanged. Repeating the test for rate-intensity function 48 h after exposure to the stressor showed no effect of prior stress on self-stimulation. This work suggests that relatively brief exposure to an uncontrollable stressor can leave a residual, 24-h depressive influence on reward system substances. Some effects of exposure to uncontrollable stressors on acquisition and maintenance of responding for rewards may be due, therefore, to attenuation of reward strength.

Restricted daily exposure to environmental enrichment increases the diversity of exploration.

The temporal parameters of environmental enrichment have been examined in two ways. The first is by limiting the total duration of environmental exposure. The effects of this procedure are well documented for both physiology and behavior. The second method is by restricting the daily exposure to environmental enrichment while keeping the days of exposure constant. The physiological effects of this procedure have been documented, but the behavioral effects which have been observed are equivocal. The purpose of the present study was to address this issue. Male rats were individually housed at 44 days of age. They were then randomly assigned to either an enriched environment or an impoverished environment. The enriched animals (EC) were exposed to the enriched environment for 2 hours daily for 30 days while the impoverished animals (IC) were handled daily but not exposed to the enriched environment. Animals were then tested on an object exploration test. The results indicate that 2 hours of daily environmental exposure is sufficient to produce object exploration effects similar to those produced by 24 hours of daily environmental exposure.

Prior exposure to stress reduces the diversity of exploratory behavior of novel objects in the rat (Rattus norvegicus).

Although a large literature has documented the varied effects of stress on an organism, relatively little attention has been devoted to investigating stress effects in ecologically relevant situations. Our experiment was conducted to assess the effects of stress on rats' (Rattus norvegicus) exploration of novel objects in a seminaturalistic and familiar environment that is relatively free of the constraints that have been placed on rats in prior investigations of stress. The results show that prior exposure to stress decreased the rats' diversity of exploration but did not affect general activity in comparison with animals not exposed to stress. We propose that the effect of stress on the qualitative aspects of exploratory behavior may be due to effects on organisms' sensitivity to predation.

Effects of Librium and shock controllability upon nociception and contextual fear.

Controllable shock is known to exert less deleterious effects than does the equivalent exposure to inescapable shock. Recent findings have encouraged speculation that some of these effects may result from differences in the severity of fear produced by the shock experiences. In particular, mediation by gamma-aminobutyric acid has been implicated. In the present experiment, we examined the possibility that chlordiazepoxide (CDP) would attenuate the impact of shock in a manner similar to that of providing control over shock. As shown by others, CDP administered prior to shock treatment blocked the long-term analgesic response, as did the provision of control during shock. Furthermore, whereas animals given controllable shock subsequently exhibited less fear of the shock context than did yoked animals, CDP treatment prior to uncontrollable shock did not appreciably reduce the contextual fear subsequently shown. These results suggest that under some conditions, controllability attenuates the impact of stress by mechanisms other than those shared by benzodiazepine treatment.

Estradiol benzoate can function as an unconditioned stimulus in a conditioned taste aversion paradigm.

The extent to which gonadal steroid hormones can serve as unconditioned stimuli in a conditioned taste aversion paradigm was examined in Rockland-Swiss albino mice. With saccharin serving as the conditioned stimulus, subcutaneously injected estradiol benzoate, but not progesterone or testosterone propionate, was found to be a potent unconditioned stimulus in both male and female mice. Dose-response effects were also observed; increasing dosages of estradiol benzoate led to increasingly stronger conditioned aversions in both males and females. The aversion detected in males was more resistant to extinction than that seen in females. Prepubertal gonadectomy reversed the sex-dependent effects of estradiol benzoate in learned aversions in adulthood; castration of males promoted the extinction process, whereas ovariectomy of females retarded extinction. The results may be useful for our understanding of the mechanisms involved in conditioned taste aversion learning as well as a wide array of hormone-dependent behavioral responses.

Exposure to low doses of the environmental chemical dieldrin causes behavioral deficits in animals prevented from coping with stress.

Experiments were conducted which assessed the effects of low doses of an environmental contaminant in conjunction with various forms of stress. Rats were given acute doses (0, 0.5, 1.5, 4.5 mg/kg) of the chemical dieldrin and subsequently exposed to a series of 40 escapable shocks, identical inescapable shocks, or no shock in an operant chamber. Eight hours later, the subjects were re-exposed in a shuttlebox to footshock which was escapable upon performance of an FR-2 shuttle response. Escape deficits which were related in magnitude to the size of the dieldrin dose were found in the inescapable shock group but not in the escapable shock or no shock groups. The data suggest that experience with the lack of control over stress is critical in determining the behavioral effects of the agent and that the behavioral effects caused by uncontrollable stress may be exacerbated by concurrent exposure to such compounds. These results are discussed in terms of previous studies on the behavioral actions of dieldrin, the response to uncontrollable stress and the common neuronal systems that may be involved.

Learned helplessness and immunization: sensitivity to response-reinforcer independence in immunized rats.

In experiments 1 and 2, we examined the learned helplessness and immunization effects using a test in which appetitive responding was extinguished by delivering noncontingent reinforcers. Contrary to learned helplessness theory, "immunized" animals showed performance virtually identical to that of animals exposed only to inescapable shock, and different from nonshocked controls. Experiment 2 suggests that the helplessness effect and the lack of immunization are not due to direct response suppression resulting from shock. In Experiment 3, where the immunization effect was assessed by measuring the acquisition of a response to obtain food when there was a positive response-reinforcer contingency, immunization was observed. These results cannot be explained on the basis of proactive interference, but suggest that animals exposed to the immunization procedure acquire an expectancy of response-reinforcer independence during inescapable shock. Thus, immunization effects may reflect the differential expression of expectancies, rather than their differential acquisition as learned helplessness theory postulates.

Uncontrollable shock proactively increases sensitivity to response-reinforcer independence in rats.

Learned helplessness theory predicts that animals exposed to inescapable shock acquire an expectancy of response-reinforcer independence, which proactively interferes with learning of response-reinforcer dependence. The theory also predicts that this expectancy can increase sensitivity to subsequent instances of response-reinforcer independence. These experiments test the latter prediction in a paradigm that minimizes the confounding effects of shock-induced activity deficits. Rats were trained to respond for food, then given either escapable, inescapable, or no shock. Subsequently, they received two sessions of response-contingent food followed by sessions of noncontingent food deliveries. During this phase, inescapably shocked animals decreased responding faster than did controls. Experiment 2 replicated this finding with a different schedule of food delivery and a procedure that more directly minimized the possibility that the outcome is due to either direct or indirect shock-induced activity changes. These results support the prediction that uncontrollable aversive events can increase an animal's sensitivity to noncontingent response-reinforcer relationships.

The role of associative factors in tolerance to the hypothermic effects of morphine in mice.

Associative learning theories of drug tolerance emphasize the importance of stimuli which predict drug administration. One such model holds that drug tolerance is due to the development of a conditional response (CR) which is directionally opposed to the unconditional response (UCR) to the drug. By virtue of their opposing natures, the overlapping occurrence of CR and UCR is seen as a diminished response, i.e., tolerance. The present experiments tested the predictions of this model using two doses of morphine, and included truly random controls to examine the role of excitatory and inhibitory conditioning in tolerance. Tolerance was greatest in mice administered morphine in the context of stimuli previously paired with drug administration, intermediate in random controls, and least or absent in mice administered the drug in the presence of cues paired with vehicle injections. No direct evidence of a compensatory CR which could offset morphine's hypothermic effect was obtained in placebo test sessions, nor was evidence for such a response obtained in cross-drug tests with amphetamine and apomorphine.

Evidence for a serotonergic mechanism of the learned helplessness phenomenon.

The present experiments examined the role of the serotonergic system in the learned helplessness phenomenon. In Experiment 1, a 200 mg/kg dose of 1-tryptophan injected 30 min prior to testing disrupted acquisition of Fixed Ratio 2 shuttle escape behavior. In Experiment 2, a 100 mg/kg dose of 5-HTP produced interference with the acquisition of the escape response. Furthermore, this interference was prevented by treatment with the serotonergic antagonist methysergide. In Experiment 3, animals were pretreated with a subeffective dose of 1-tryptophan in combination with subeffective exposure to inescapable shock. These animals showed a deficit in the acquisition of FR-2 shuttle escape. In Experiment 4, combined exposure to a subeffective dose of 5-HTP and inescapable shock (40 trials) resulted in an acquisition deficit. This deficit was reversed by methysergide. Experiment 5 showed that the detrimental effects of exposure to prolonged (80 trials) of inescapable shock can be prevented by treatment with methysergide. These studies implicate the serotonergic system as a possible mediator of the learned helplessness phenomenon.

Cross-motivational effects of inescapable shock are associative in nature.

Exposure to inescapable shock interferes with the subsequent acquisition of an appetitive operant. This deficit may be due to either associative interference or activity reduction from the inescapable shock pretreatment. The relative importance of these two factors was examined by using an appetitive response choice discrimination procedure and concurrently measuring activity. Experiment 1 demonstrated separate associative and activity effects of inescapable shock, in that the animals exposed to inescapable shock made more incorrect responses than controls and were lower in activity. Experiment 2 demonstrated that these effects resulted from the uncontrollability of the shock, not from shock exposure per se. In Experiment 3, residual effects of inescapable shock were investigated by exposing animals to discrimination reversals. On these tests, inescapably shocked animals showed performance inferior to nonshocked controls, a result indicating that the effects of inescapable shock are not completely reversed by experience with contingent reward in the discrimination task. No evidence of an activity deficit was observed during this discrimination reversal. These results strongly suggest that associative factors play a more important role than activity reduction in mediating the effects of inescapable shock, at least when these are measured in an appetitive context.