Modeling nicotine addiction in rats.

Among the human population, 15% of drug users develop a pathological drug addiction. This figure increases substantially with nicotine, whereby more than 30% of those who try smoking develop a nicotine addiction. Drug addiction is characterized by compulsive drug-seeking and drug-taking behaviors (craving), and loss of control over intake despite impairment in health, social, and occupational functions. This behavior can be accurately modeled in the rat using an intravenous self-administration (IVSA) paradigm. Initial attempts at establishing nicotine self-administration had been problematic, yet in recent times increasingly reliable models of nicotine self-administration have been developed. The present article reviews different characteristics of the nicotine IVSA model that has been developed to examine nicotine reinforcing and motivational properties in rats.

Repeated episodes of heroin cause enduring alterations of circadian activity in protracted abstinence.

Opiate withdrawal is followed by a protracted abstinence syndrome consisting of craving and physiological changes. However, few studies have been dedicated to both the characterization and understanding of these long-term alterations in post-dependent subjects. The aim of the present study was to develop an opiate dependence model, which induces long-lasting behavioral changes in abstinent rats. Here, we first compared the effects of several protocols for the induction of opiate dependence (morphine pellets, repeated morphine or heroin injections) on the subsequent response to heroin challenges (0.25 mg/kg) at different time points during abstinence (3, 6, 9 and 18 weeks). In a second set of experiments, rats were exposed to increasing doses of heroin and subsequently monitored for general circadian activity up to 20 weeks of abstinence. Results show that heroin injections rather than the other methods of opiate administration have long-term consequences on rats' sensitivity to heroin with its psychostimulant effects persisting up to 18 weeks of abstinence. Moreover, intermittent episodes of heroin dependence rather than a single exposure produce enduring alteration of the basal circadian activity both upon heroin cessation and protracted abstinence. Altogether, these findings suggest that the induction of heroin dependence through intermittent increasing heroin injections is the optimal method to model long-term behavioral alterations during protracted abstinence in rats. This animal model would be useful in further characterizing long-lasting changes in post-dependent subjects to help understand the prolonged vulnerability to relapse.

The addition of five minor tobacco alkaloids increases nicotine-induced hyperactivity, sensitization and intravenous self-administration in rats.

Several minor tobacco alkaloids have been found to exhibit properties pharmacologically relevant to the addictive profile of tobacco; however, little is known of their effects on a behavioural model of drug addiction. In this study we compared the locomotor and reinforcing effects of intravenous nicotine (30 microg/kg per infusion) vs. a cocktail of nicotine plus five minor alkaloids found in tobacco smoke (anabasine, nornicotine, anatabine, cotinine and myosmine). Rats were initially tested for their locomotor response to nicotine or nicotine plus the minor alkaloids with six intravenous injections over 1 h. We then assessed the spontaneous acquisition of intravenous self-administration with nicotine or nicotine plus the minor alkaloids, under a fixed-ratio 1 schedule followed by responding on a fixed-ratio 5 schedule, progressive-ratio schedule and a single within-session ascending dose-response test. The activity test was repeated following the progressive-ratio phase to assess locomotor sensitization. A second group of rats were then tested on the locomotor procedure to better clarify the role of each individual minor alkaloid in nicotine-induced locomotor activity. Compared to nicotine alone, addition of the minor tobacco alkaloids increased locomotor activity and increased locomotor sensitization following self-administration. During fixed-ratio 5, progressive ratio and the dose-response test, rats receiving nicotine plus the minor alkaloids responded significantly more than those receiving nicotine alone. Testing of each minor alkaloid in the second experiment indicated that anatabine, cotinine and myosmine individually increased nicotine-induced locomotor activity. These results suggest that the minor tobacco alkaloids, particularly anatabine, cotinine and myosmine, may increase the motivation for nicotine and thus facilitate smoking behaviour.

Monoamine oxidase inhibition dramatically prolongs the duration of nicotine withdrawal-induced place aversion.

BACKGROUND: Long-lasting effects of withdrawal from nicotine are hypothesized to contribute to relapse and persistence of tobacco habits, and significant evidence supports a role of monoamine oxidase inhibitors (MAOI) contained in cigarette smoke as potent modulators of the rewarding effects of tobacco. METHODS: With quantification of somatic signs of withdrawal and the place aversion conditioning paradigm, we assessed the effects of MAOI pretreatment on both somatic and aversive motivational components of mecamylamine-induced nicotine withdrawal in rats rendered dependent on nicotine by the subcutaneous implantation of osmotic minipumps (vehicle or nicotine tartrate 9 mg/kg/day). RESULTS: In nicotine-infused rats, mecamylamine induced a place aversion that lasted 6 weeks. When nicotine-infused rats were also treated with a MAOI, mecamylamine-induced conditioned place aversion persisted for at least 8 months of abstinence. The MAOI treatment slightly decreased ratings of somatic signs induced by mecamylamine administration but had no effect on the threshold or the magnitude of mecamylamine-induced conditioned place aversion. CONCLUSIONS: These results show that MAOI pretreatment induces a long-lasting conditioned placed aversion associated with nicotine withdrawal, possibly through a potentiation of learning and memory process, and provides some indications on protracted abstinence that might be useful for delineating the neurobiological substrate of relapse.

High-efficacy 5-hydroxytryptamine 1A receptor activation counteracts opioid hyperallodynia and affective conditioning.

Pain may become intractable as tolerance develops to opioids and the opioids, paradoxically, induce pain. We examined the hypothesis that the analgesia produced by the novel analgesic and high-efficacy 5-hydroxytryptamine (5-HT)(1A) receptor agonist (3-chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl)-amino]methyl]piperidin-1-yl]methanone, fumaric acid salt (F 13640) may counteract opioid-induced pain. In studies of the somatosensory quality of pain in infraorbital nerve-injured rats, morphine infusion (5 mg/day) by means of osmotic pumps initially caused analgesia (i.e., decreased the behavioral response to von Frey filament stimulation), followed by hyperallodynia and analgesic tolerance. Infusion of F 13640 (0.63 mg/day) prevented the development of opioid hyperallodynia and reversed opioid hyperallodynia once established. In studies of the affective/motivational quality of pain, F 13640 both prevented and reversed the conditioned place aversion induced by naloxone (0.04 mg/kg i.p.) in morphine-infused rats; F 13640 also prevented and reversed the conditioned place preference induced by morphine injections (7.5 mg/kg i.p.). The data confirm that opioids produce bidirectional hypo- and proalgesic actions, and offer initial evidence that high-efficacy 5-HT(1A) receptor activation counteracts both the sensory and the affective/motivational qualities of opioid-induced pain. The data also indicate that F 13640 may be effective with opioid-resistant pain. It further is suggested that opioid addiction may represent self-therapy of opioid-induced pathological pain.

Monoamine oxidase A rather than monoamine oxidase B inhibition increases nicotine reinforcement in rats.

Although nicotine is considered to be responsible for the addictive properties of tobacco, growing evidence underlines the importance of non-nicotine components in smoking reinforcement. It has been shown that tobacco smoke contains monoamine oxidase (MAO) A and B inhibitors and decreases MAO-A and MAO-B activity in smokers. Here, we investigated the effects of clorgyline hydrochloride (irreversible MAO-A inhibitor; 2 mg/kg/day), selegiline (irreversible MAO-B inhibitor; 4 mg/kg) and the beta-carboline norharmane hydrochloride (reversible MAO-B inhibitor; 5 mg/kg/day) treatments on nicotine self-administration (30 microg/kg/infusion, free base) in rats. Independent of the responsiveness to novelty and locomotor activity stimulation, only clorgyline hydrochloride treatment increased the intake of nicotine in a fixed-ratio schedule (FR5) of reinforcement. When a progressive-ratio schedule was implemented, both clorgyline hydrochloride and norharmane hydrochloride treatments potentiated the reinforcing effects of nicotine, whereas selegiline had no effect. Taken together, these results indicate that MAO-A inhibition interacts with nicotine to enhance its rewarding effects in rats and suggest that other compounds present in tobacco, such as beta-carboline, may also play an important role in sustaining smoking behavior in humans.

Monoamine oxidase inhibition dramatically increases the motivation to self-administer nicotine in rats.

Nicotine is the major neuroactive compound of tobacco, which has, by itself, weak reinforcing properties. It is known that levels of the enzymes monoamine oxidase A (MAO-A) and MAO-B are reduced in the platelets and brains of smokers and that substances, other than nicotine, present in tobacco smoke have MAO-inhibitory activities. Here, we report that inhibition of MAO dramatically and specifically increases the motivation to self-administer nicotine in rats. These effects were more prominent in rats selected for high responsiveness to novelty than in rats with low responsiveness to novelty. The results suggest that the inhibition of MAO activity by compounds present in tobacco smoke may combine with nicotine to produce the intense reinforcing properties of cigarette smoking that lead to addiction.

Mesolimbic dopamine drives the diurnal variation in opiate-induced feeding.

Brain opioid peptides modulate feeding behavior and opiate drugs have powerful orexigenic effects in mammals. Recent studies have shown that opiate-induced eating depends, though not exclusively, on mu-opioid receptors located in the ventral striatum. Here we report that morphine orexigenic effects vary with the time of day according to a biphasic pattern. The effects first increase and then decrease during the light phase, with the peak effect occurring in the middle of this phase. This diurnal profile is shifted toward the dark phase after dopamine deafferentation of the ventral striatum. Consequently, the peak effect of morphine is delayed and occurs just before the dark phase. This finding suggests that mesolimbic dopamine transmission contributes to the neural mechanisms that normally drive the circadian timing of opioid-dependent feeding.

A specific limbic circuit underlies opiate withdrawal memories.

Compulsive drug-seeking behavior and its renewal in former drug addicts is promoted by several situations, among which reactivation of drug withdrawal memories plays a crucial role. A neural hypothesis is that such memories reactivate the circuits involved in withdrawal itself and promote a motivational state leading to drug seeking or taking. To test this hypothesis, we have analyzed the neural circuits and cell populations recruited when opiate-dependent rats are reexposed to stimuli previously paired with withdrawal (memory retrieval) and compared them with those underlying acute withdrawal during conditioning (memory formation). Using in situ hybridization for c-fos expression, we report here that reexposure to a withdrawal-paired environment induced conditioned c-fos responses in a specific limbic circuit, which can be partially dissociated from the structures involved in acute withdrawal. At the amygdala level, c-fos responses were doubly dissociated between the central and basolateral (BLA) nuclei, when comparing the two situations. Detailed phenotypical analyses in the amygdala and ventral tegmental area (VTA) show that specific subpopulations in the BLA are differentially involved in the formation and retrieval of withdrawal memories, and strikingly that a population of VTA dopamine neurons is activated in both situations. Together, this indicates that withdrawal memories can drive activity changes in specific neuronal populations of interconnected limbic areas known to be involved in aversive motivational processes. This first study on the neural substrates of withdrawal memories strongly supports an incentive-motivational view of withdrawal in opiate addiction that could be crucial in compulsive drug seeking and relapse.

Buprenorphine and a CRF1 antagonist block the acquisition of opiate withdrawal-induced conditioned place aversion in rats.

Conditioned place aversion in rats has face validity as a measure of the aversive stimulus effects of opiate withdrawal that reflects an important motivational component of opiate dependence. The purpose of the present study was to validate conditioned place aversion as sensitive to medications that will alleviate the aversive stimulus effects of opiate withdrawal in humans, and to extend this model to the exploration of the neuropharmacological basis of the motivational effects of opiate withdrawal. Male Sprague-Dawley rats were implanted with two subcutaneous morphine pellets and 5 days later began place conditioning training following subcutaneous administration of a low dose of naloxone. Animals were subjected to three pairings of a low dose of naloxone (15 microg/kg, s.c.) to one arm of a three-chambered place conditioning apparatus. Buprenorphine administered prior to each pairing dose-dependently blocked the place aversion produced by precipitated opiate withdrawal. A corticotropin-releasing factor-1 (CRF1) receptor antagonist (antalarmin) also reversed the place aversion produced by precipitated opiate withdrawal. Antalarmin did not produce a place preference or place aversion by itself in morphine-dependent rats. No effect was observed with pretreatment of the dopamine partial agonist terguride or the selective serotonin reuptake inhibitor fluoxetine. Also, chronic pretreatment with acamprosate (a glutamate receptor modulator used to prevent relapse in alcohol dependence) did not alter naloxone-induced place aversion. Buprenorphine by itself in dependent rats produced a mild place preference at low doses and a mild place aversion at higher doses. These results suggest that buprenorphine blocks the aversive stimulus effects of precipitated opiate withdrawal in rats and provides some validity for the use of place conditioning as a measure that is sensitive to potential opiate-dependence medications. In addition, these results suggest that CRF1 antagonists can block the aversive stimulus effects of opiate withdrawal and may be potential therapeutic targets for opiate dependence.

Changes in dopaminergic neurotransmission do not alter somatic or motivational opiate withdrawal-induced symptoms in rats.

Opiate withdrawal has been correlated with decreased extracellular dopamine (DA) levels in the nucleus accumbens (NAC) of morphine-dependent rats. The authors tested the hypothesis that DA transmission plays a critical role in the induction of motivational and somatic withdrawal symptoms. First, the authors used a 6-hydroxydopamine-induced lesion of the NAC to chronically disrupt mesolimbic DA transmission. Second, global DA neurotransmission was acutely stimulated by the nonselective DA agonist (apomorphine) or inhibited by nonselective DA antagonists (droperidol or flupentixol). Morphine-dependent rats bearing 6-hydroxydopamine-induced lesions displayed naloxone-precipitated motivational and somatic withdrawal symptoms similar to those of sham-lesioned rats. Administration of apomorphine did not reduce naloxone-induced opiate withdrawal. Moreover, in total absence of naloxone, DA antagonists did not precipitate either conditioned place aversion or somatic abstinence signs in dependent rats. Taken together, these findings suggested that DA transmission is not critical for the induction of opiate withdrawal syndrome.

Chronic interferon-alpha potentiates latent inhibition in rats.

Several recent reports based on microdialysis have shown that repeated injections of IFN-alpha induce reduced dopamine (DA) activity in the rodent brain, and specifically within the striatum. This mesolimbic DA system is thought to play a central role in controlling latent inhibition (LI), which refers to the phenomenon in which conditioning proceeds more slowly to an irrelevant pre-exposed (PE) stimulus. The purpose of the present experiment was to evaluate whether chronic treatment with IFN-alpha may induce similar effects on LI as other DA-depleting manipulations. Rats were injected daily, for 24 days either with 10(4)IU IFN-alpha or vehicle, while monitoring their locomotor activity in photocell cages. No changes in locomotor activity, either during the acute period after the first injection, or progressively over the chronic period were observed. LI was then evaluated in these rats with a Conditioned Taste Aversion (CTA) paradigm. Half of each group of rats were pre-exposed to the 5% sucrose Conditioned Stimulus (CS) for 30 min on three successive days before the conditioning session in which the 30 min access to the 5% sucrose solution was followed by a 0.15M injection of 127 mg/kg LiCl. Two test sessions, in which rats were given a choice between the 5% sucrose solution and water, were given 48 and 72 h after the conditioning session. The results showed moderate LI in the control group, which was considerably potentiated in the IFN-alpha group despite the absence of any change in locomotor activity. These results are interpreted as providing indirect support for the theory that suggests that LI depends on DA activity in the nucleus accumbens (NAcc).

Inhibition of 5-HT neurotransmission increases clonidine protective effects on naloxone-induced conditioned place aversion in morphine-dependent rats.

Previous pharmacological studies have implicated serotonergic brain systems in opiate-withdrawal-precipitated conditioned place aversion. To assess this hypothesis, we tested the effects of either (i). a near-total 5,7-dihydroxytryptamine-induced lesion (90% depletion) or (ii). an acute serotonin (5-HT) inhibition induced by the specific stimulation of 5-HT1A autoreceptors (8-OHDPAT 5-100 microg/kg), on naloxone-induced conditioned place aversion in morphine-dependent rats. Morphine dependence was induced by the implantation of morphine slow-release pellets. The protective properties of clonidine (an alpha-2 adrenergic agonist classically given for opiate detoxification) were also tested after inhibition of 5-HT transmission. Serotonergic lesions in morphine-dependent rats failed to alter naloxone-induced conditioned place aversion but increased the sensitivity to the protective effects of clonidine. Acute neuropharmacological blockade of serotonin transmission also potentiated the clonidine effects on naloxone-induced conditioned place aversion. When combined with the 5-HT1A agonist 8-OHDPAT, clonidine was also found to be more potent. Further understanding of this serotonin/noradrenaline interaction might help devise new therapeutic treatments for the acute opiate withdrawal syndrome.

In the rat forced swimming test, chronic but not subacute administration of dual 5-HT/NA antidepressant treatments may produce greater effects than selective drugs.

UNLABELLED: The rat forced swimming test (FST) distinguishes selective serotonin (5-HT) and selective noradrenaline (NA) reuptake-inhibitors, which respectively increase swimming and climbing behaviours. However, NA-system-mediated inhibition of 5-HT-induced swimming prevents dual 5-HT/NA reuptake-inhibition to produce concurrently climbing with swimming. Since adaptative neurochemical processes occur in the treatment of depression, we examined the influence of long-term antidepressant treatment on these interactions. METHODS: (1) Selective [fluoxetine: 10 mg/kg; desipramine: 10 mg/kg] and non-selective [milnacipran: 40 mg/kg; mirtazapine: 20 mg/kg] antidepressants were administered subacutely (3inj) and chronically (17inj) over 16 days. (2) A subacute fluoxetine-desipramine combination (10-10 mg/kg) was administered in rats that were pre-treated with chronic-desipramine (10 mg/kg per day, 14 days). (3) NA-system-mediated interactions were further examined by combining the alpha(2)-receptor agonist clonidine (5, 10, 20, 200 microg/kg) with 10 mg/kg fluoxetine. RESULTS: (1) Long-term treatment with either fluoxetine or desipramine does not modify the behavioural response produced by their subacute administration. (2) In contrast, whereas subacute-milnacipran increases climbing solely, chronic-milnacipran produces greater anti-immobility effects and increases both climbing and swimming behaviours. Similarly, the fluoxetine-desipramine combination produces climbing solely, but increases both climbing and swimming behaviours in animals pre-treated with chronic-desipramine. Chronic but not subacute-mirtazapine increases swimming behaviour. (3) clonidine dose-dependently antagonizes fluoxetine-induced anti-immobility effects and swimming behaviour. CONCLUSIONS: Chronic enhancement of NA-transmission alters NA-system-mediated inhibition of 5-HT-induced behaviour in the FST, which may involve alpha(2)-receptors.

Neural correlates of the motivational and somatic components of naloxone-precipitated morphine withdrawal.

In morphine-dependent rats, low naloxone doses have been shown to induce conditioned place aversion, which reflects the negative motivational component of opiate withdrawal. In contrast, higher naloxone doses are able to induce a 'full' withdrawal syndrome, including overt somatic signs. The c-fos gene is commonly used as a marker of neuronal reactivity to map the neural substrates that are recruited by various stimuli. Using in situ hybridization, we have analysed in the brain of morphine-dependent rats the effects of acute withdrawal syndrome precipitated by increasing naloxone doses on c-fos mRNA expression. Morphine dependence was induced by subcutaneous implantation of slow-release morphine pellets for 6 days and withdrawal was precipitated by increasing naloxone doses inducing the motivational (7.5 and 15 micro g/kg) and somatic (30 and 120 micro g/kg) components of withdrawal. Our mapping study revealed a dissociation between a set of brain structures (extended amygdala, lateral septal nucleus, basolateral amygdala and field CA1 of the hippocampus) which exhibited c-fos mRNA dose-dependent variations from the lowest naloxone doses, and many other structures (dopaminergic and noradrenergic nuclei, motor striatal areas, hypothalamic nuclei and periaqueductal grey) which were less sensitive and recruited only by the higher doses. In addition, we found opposite dose-dependent variations of c-fos gene expression within the central (increase) and the basolateral (decrease) amygdala after acute morphine withdrawal. Altogether, these results emphasize that limbic structures of the extended amygdala along with the lateral septal nucleus, the basolateral amygdala and CA1 could specifically mediate the negative motivational component of opiate withdrawal.

Dorsal and median raphe serotonergic system lesion does not alter the opiate withdrawal syndrome.

Previous pharmacological studies have implicated serotonergic brain systems in opiate withdrawal. To test the hypothesis that serotonin (5-HT) has a critical role in the development of opiate withdrawal, we have employed a near-total brain 5-HT system lesion technique (90% depletion) using 5,7-dihydroxytryptamine combined with induction of opiate dependence by implantation of morphine pellets or by repeated injections of increasing doses of morphine. The effects of serotonergic neuron lesion were examined on spontaneous opiate withdrawal (changes in circadian locomotor activity) and naloxone-precipitated opiate withdrawal syndrome (the somatic aspect). The antiwithdrawal properties of clonidine, an alpha(2)-adrenoceptor agonist currently used for clinical treatment for the somatic signs of opiate withdrawal, were tested also in the lesioned rats. Our findings show that serotonergic lesions in morphine-dependent rats did not alter either the spontaneous or the naloxone-induced withdrawal syndrome (with exception of jumping behavior). Moreover, clonidine alleviated the naloxone-induced withdrawal syndrome in lesioned as well as in sham-operated morphine-dependent rats. These results demonstrate that 5-HT systems are not directly responsible for the development of the somatic opiate withdrawal syndrome in morphine-dependent rats.

In the rat forced swimming test, NA-system mediated interactions may prevent the 5-HT properties of some subacute antidepressant treatments being expressed.

In the rat forced swimming test (FST), reuptake inhibitors selective of either serotonin (5-HT) or noradrenaline (NA) decrease immobility duration, and increase, respectively, swimming and climbing behaviour. In this study, an almost total 6-OHDA-induced NA-depletion prevented the behavioural effects of desipramine, but not fluoxetine. Interestingly, the serotonin/noradrenaline-reuptake-inhibitor milnacipran, as well as a (desipramine+fluoxetine) combination, could produce both swimming and climbing behaviour in NA-lesioned rats, but not in non-lesioned. The new antidepressant mirtazapine, which enhances both 5-HT and NA transmissions, supposedly through the antagonizing of alpha(2)-adrenoreceptors, dose-dependently reduced immobility and increased climbing behaviour. Interestingly, a (mirtazapine+fluoxetine) combination treatment resulted in additive anti-immobility effects and in the summation of fluoxetine-induced swimming with mirtazapine-induced climbing. Taken together, these data suggest that the NA system mediates presynaptic inhibiting interactions on the 5-HT system, that may involve alpha(2)-receptors, and that may limit the efficacy of mixed serotonin/noradrenaline reuptake inhibition in subacute antidepressant treatments.

Central 5-HT(4) receptors and dopamine-dependent motor behaviors: searching for a functional role.

In this study, we evaluated the role of central 5-HT(4) receptors in the control of motor behaviors related to change of nigrostriatal dopamine (DA) transmission, namely, stereotyped behavior and catalepsy in rats. Indeed, given that 5-HT(4) receptors indirectly modulate nigrostriatal DA neuron activity, we hypothesized that these receptors would regulate nigrostriatal DA transmission in the basal ganglia, and consequently, associated motor responses. Stereotypy was induced either by an acute administration of apomorphine (0.3 and 1.5 mg/kg sc), or by a single morphine administration (15 mg/kg sc) in chronically morphine-treated (15 mg/kg sc, twice daily for 10 days) rats. Catalepsy was induced by the typical neuroleptic haloperidol (HAL; 1 mg/kg sc). The selective 5-HT(4) antagonist, GR 125487 (1 mg/kg ip), modified neither apomorphine- nor morphine-induced stereotypy. HAL-induced catalepsy, while reduced by the systemic administration of the 5-HT(1A) agonist 8-OH-DPAT (0.1 mg/kg sc), was insensitive to GR 125487, systemically (1, 3, 10 mg/kg ip) or locally (20 and 40 nmol/20 microl) administered into the third ventricle. Also, HAL-induced catalepsy was not affected by the selective 5-HT(4) antagonist GR 113808 (3 mg/kg ip). The obtained results indicate that 5-HT(4) receptor antagonism does not modulate motor behaviors related to change of striatal DA transmission.

Ethological analysis of morphine withdrawal with different dependence programs in male mice.

This work was performed to clarify the differences between a long or short development of morphine dependence as well as between a recently installed or a long-term dependence. Morphine withdrawal in rats is a well-characterized phenomenon but this is not so in mice. A study of the principal withdrawal signs have been performed in mice, evaluating their specificity and particular profile of appearance in each type of dependence. Mice were divided into two groups that received increasing doses of morphine every 24 h, three groups that received increasing doses of morphine twice a day for 3 days, and a control group that received saline. Naloxone-induced opiate withdrawal was evaluated following short-term exposition to morphine [Test 1 (T1)--saline and Test 2 (T2)--naloxone] and long-term exposition to morphine [Test 3 (T3)--naloxone and Test 4 (T4)--saline]. Morphine administration twice a day is more effective in inducing opiate dependence than once a day, and with the latter, the duration of morphine exposure increases the intensity of withdrawal signs. Weight loss, diarrhea, body shakes, jumping, paw tremor, ptosis, piloerection, and the modified Gellert-Holtzman scale for mice are specific patterns of naloxone-induced withdrawal. The first four signs allow the discrimination between different levels of opiate dependence. Body care, piloerection, and the modified Gellert-Holtzman scale could be useful to detect conditioned withdrawal.