Physical Dependence In Rats Research Articles

Blocking IL-17A prevents oxycodone-induced depression-like effects and elevation of IL-6 levels in the ventral tegmental area and reduces oxycodone-derived physical dependence in rats

Oxycodone is the most prescribed opioid for pain management and has been available in clinics for almost a century, but effects of chronic oxycodone have been studied less than morphine in preclinical and clinical studies. Newly developed depression has been coupled with chronic oxycodone use in a few clinical studies, but no preclinical studies have investigated the pathogenesis of oxycodone-induced depression. Gut microbiome changes following oxycodone use is an understudied area, and interleukin-17A (IL-17A) is linked to both the development of mood disorders and regulation of gut microbiome. The present study investigated effects of chronic oxycodone exposure on mood-related behaviors (depression and anxiety), pain hypersensitivity, physical dependence, immune markers, and the gut microbiome and tested the hypothesis that blocking IL-17A with a systemically administered monoclonal antibody reduces oxycodone-derived effects. Oxycodone (using an incremental dosing regimen) or saline was injected twice a day for 12 days. IL-17A Ab (200 µg/100 µl) or saline was administered every 3rd day during the 12-day interval. Chronic oxycodone induced a depression-like effect, but not anxiogenic- or anxiolytic-like effects; promoted hyperalgesia; increased IL-17A and IL-6 levels in the ventral tegmental area (VTA); and induced physical dependence. IL-17A Ab co-administration with oxycodone prevented the depression-like effect and hyperalgesia, reduced naloxone-precipitated withdrawal signs, and normalized the increase in cytokine levels. Chronic oxycodone exposure did not affect gut microbiome and integrity. Our results identify a role for IL-17A in oxycodone-related behavioral and neuroimmune effects and show that IL-17A Ab has potential therapeutic value in blocking these effects. Given that humanized IL-17A Ab is approved for treatment of psoriasis and psoriatic arthritis, our findings point toward studying it for use in the treatment of oxycodone use disorder.

Opioid receptors and butorphanol dependence

Butorphanol, a mixed opioid agonist/antagonist, is considered to be a relatively safe drug when used within the therapeutic dose range. However, diversional uses of butorphanol involving high dose have been documented. In the present review, the relative involvement of μ, δ, and κ-opioid receptors in butorphanol physical dependence in rats is discussed. Physical dependence was produced by continuous intracerebroventricular (icv) infusion of butorphanol (26 nmol/h) for 72 h in male Sprague-Dawley rats. Multiple icv injections of β-funaltrexamine (β-FNA, a μ-antagonist, 12, 24, or 48 nmol/5 μl), naltrindole (NTI, a δ-antagonist, 0.1, 1, or 10 nmol/5 μl), or nor-binaltorphimine (nor-BNI, a κ-antagonist, 12, 24, or 48 nmol/5 μl) significantly attenuated the development of butorphanol dependence. Furthermore, icv administration of both NTI (24, 48, or 100 nmol/5 μl) and nor-BNI (3, 10, 20, 48 or 100 nmol/5 μl) precipitated withdrawal behaviors, whereas, β-FNA (12, 24, 48, or 100 nmol/5 μl) was unable to elicit withdrawal signs in butorphanol-dependent rats. The results indicate that the development of butorphanol dependence is due to effects of butorphanol on central μ-, δ-, and κ-opioid receptors.

Honey potentially mitigates morphine analgesic tolerance and physical dependence in rats

Introduction:Honey has been used traditionally in medicine as well as food supplements. Honeybees are said to be able to cure many diseases. However, its influences on opioid tolerance and dependence have not yet been clarified.Materials and Methods:Adult male Sprague- Dawley rats were rendered tolerant to the analgesic effect of morphine by injection of morphine (10 mg/kg, i.p.) twice daily for 14 days. To develop morphine dependence rats given escalating doses of chronic morphine. To determine the effect of stingless bee honey on the development of morphine tolerance and dependence. The hotplate and naloxone precipitation tests were used to assess the degree of tolerance and dependence, respectively.The results: Our results showed that chronic morphine-injected rats displayed tolerance to the analgesic effect of morphine as well as morphine dependence. Methadone+morphine (MetM), methadone+morphine+ honey (MetMH) and morphine+Honey (MH) significantlylower the development of morphine tolerance with p-value p<0.05. In addition, concomitant treatment of morphine with MH and MetMH attenuated almost all of the naloxone-induced withdrawal signs which include abdominal contraction, diarrhea, pertussis, teeth chattering, and jumping.Conclusion: The data indicate that honey has a potential to reduce tolerant and dependence property.Bangladesh Journal of Medical Science Vol.17(1) 2018 p.138-143

Blockade of orexin type-1 receptors in locus coeruleus nucleus attenuates the development of morphine dependency in rats

The aim of this study was to evaluate the effects of orexin type-1 receptor (OX1R) antagonism in locus coeruleus (LC) nucleus on the development of morphine physical dependence in rats. Animals were rendered dependent on morphine by subcutaneous (s.c.) administration of morphine sulfate (6, 16, 26, 36, 46, 56 and 66mg/kg, 2ml/kg) at set intervals of 24h for 7 days. Immediately before each morphine administration, the animals received intra-LC administration of SB-334867 (3mM, 0.2μl), a selective orexin type-1 receptor antagonist. On day 8, naloxone (3mg/kg, i.p.) was injected and physical dependence was evaluated for 30min. Our results showed that administration of OX1R antagonist before each morphine injection significantly decreased somatic signs of naloxone-induced morphine withdrawal syndrome, including defecation, wet-dog shake, diarrhea, jumping, scratching, and teeth chattering. These results suggest that the activation of OX1R in LC nucleus might be involved in the development of morphine dependency.

Repeated central administration of selegiline attenuated morphine physical dependence in rat

BackgroundLong-term exposure to opiates induces physical dependence; however, the neurobiological mechanisms of this phenomenon are not completely clear. The purpose of this study was to evaluate the effects of systemic and intracerebroventricular (icv) administration of selegiline (a selective inhibitor of monoamine oxidase B) on the morphine withdrawal syndrome in rats. MethodsTo this aim, adult male Sprague Dawley rats were selected randomly, and then growing doses of morphine were administered subcutaneously at an interval of 12h for nine days with the intention of inducing dependency. Nine days after, only the morning dose of morphine was administered, followed by systemic or central injection of saline or selegiline. Later, naloxone was injected after 30min and withdrawal signs recorded for a period of 60min. ResultsResults showed failure of systemic administration of selegiline in changing the withdrawal symptoms; nevertheless, icv injection attenuated the withdrawal signs significantly. ConclusionIn conclusion we found that central administration of selegiline attenuated morphine withdrawal symptoms

Extracellular Signal-Regulated Kinase 5 in the Cerebrospinal Fluid-Contacting Nucleus Contributes to Morphine Physical Dependence in Rats

The cerebrospinal fluid-contacting nucleus (CSF-CN) may influence actual composition of the CSF for non-synaptic signal transmission via releasing or absorbing bioactive substances, which distributes and localizes in the ventral periaqueductal central gray of the brainstem. Previous studies demonstrated that CSF-CN was involved in neuropathic pain and morphine dependence. Thus, to identify whether extracellular signal-regulated kinase 5 (ERK5) distributed in the CSF-CN and its function on the formation and development of morphine physical dependence, morphine withdrawal-like behavioral test and immunofluorescent technique were used in this research. Morphine was subcutaneously injected by an intermittent and escalating procedure to induce physical dependence, which was measured by withdrawal symptoms. In this study, we found that horseradish peroxidase-conjugated toxin subunit B/p-ERK5 double-labeled neurons expressed in the CSF-CN of normal rats. ERK5 signaling pathway was remarkably activated by naloxone-precipitated withdrawal in the CSF-CN. Moreover, selective attenuation of p-ERK5 expression in the CSF-CN by lateral ventricle injection of BIX02188 could significantly relieve morphine withdrawal symptom. These findings confirmed that the activation of p-ERK5 in the CSF-CN might contribute to morphine physical dependence.

Long-term daily access to alcohol alters dopamine-related synthesis and signaling proteins in the rat striatum

Chronic alcohol exposure can adversely affect neuronal morphology, synaptic architecture and associated neuroplasticity. However, the effects of moderate levels of long-term alcohol intake on the brain are a matter of debate. The current study used 2-DE (two-dimensional gel electrophoresis) proteomics to examine proteomic changes in the striatum of male Wistar rats after 8months of continuous access to a standard off-the-shelf beer in their home cages. Alcohol intake under group-housed conditions during this time was around 3–4g/kg/day, a level below that known to induce physical dependence in rats. After 8months of access rats were euthanased and 2-DE proteomic analysis of the striatum was conducted. A total of 28 striatal proteins were significantly altered in the beer drinking rats relative to controls. Strikingly, many of these were dopamine (DA)-related proteins, including tyrosine hydroxylase (an enzyme of DA biosynthesis), pyridoxal phosphate phosphatase (a co-enzyme in DA biosynthesis), DA and cAMP regulating phosphoprotein (a regulator of DA receptors and transporters), protein phosphatase 1 (a signaling protein) and nitric oxide synthase (which modulates DA uptake). Selected protein expression changes were verified using Western blotting. We conclude that long-term moderate alcohol consumption is associated with substantial alterations in the rat striatal proteome, particularly with regard to dopaminergic signaling pathways. This provides potentially important evidence of major neuroadaptations in dopamine systems with daily alcohol consumption at relatively modest levels.

Blockade of orexin receptor 1 attenuates the development of morphine tolerance and physical dependence in rats

The goals of this study were to evaluate the effects of pretreatment by orexin receptor-1 antagonist on the development of morphine tolerance and physical dependence in rat. Animals were rendered dependent on morphine by subcutaneous (SC) injection of morphine sulfate (10mg/kg) at set intervals of 12h for 10days. Just before the morphine administration, the animals received SB-334867, a selective orexin receptor 1 (OXR1) antagonist. To assess morphine tolerance, the antinociceptive responses of morphine were measured using the warm-water tail immersion test before and after its administration. On day 11, naloxone was injected 2h after morphine administration and the physical dependence evaluated by quantifying/scoring naloxone-precipitated withdrawal signs for 30min. The effect of chronic SB-334867 on locomotion was carried out by calculating the number of grid crossings as a measure of locomotor activity. Our findings demonstrated that although morphine-tolerance tended to develop in response to repeated injections of morphine, pre-treatment of OXR1 antagonist prevented this effect, causing a delay in the development of morphine-tolerance. Moreover, co-administration of orexin receptor 1 antagonist with morphine significantly decreased the somatic signs of withdrawal including diarrhea, teeth chattering, jumping, and defecation. Administration of SB-334867 alone or in a chronic co-administration with morphine failed to change locomotor activity. These results suggest that the activation of OXR1 might be involved in the development of morphine tolerance and dependence.

Activation of the spinal extracellular signal-regulated kinase 5 signaling pathway contributes to morphine physical dependence in rats

The activation of mitogen-activated protein kinases (MAPKs) has been observed in synaptic plasticity processes of learning and memory in morphine dependence. However, the role of extracellular signal-regulated protein kinase 5 (ERK5), a member of MAPKs, has not been studied yet in morphine dependence. To identify the function of ERK5 in the formation and development of morphine physical dependence, morphine withdrawal-like behavioral test and western blot technique were used in this research. Morphine was subcutaneously injected by an intermittent and escalating procedure to induce physical dependence, which was measured by withdrawal symptoms. In this study, spinal ERK5 signaling pathway was remarkably activated by chronic morphine injection and naloxone-precipitated withdrawal. Intrathecal injection of BIX02188, a novel specific inhibitor of mitogen-activated protein kinases kinase 5 (MEK5), produced a dose- and time-dependent inhibition of the activation of spinal ERK5, without affecting activation of other MAPKs. Moreover, selective attenuation of spinal p-ERK5 expression by BIX02188 could significantly relieve morphine withdrawal symptom, accompanying with the decreased phosphorylation of cAMP response-element binding protein (CREB) in the spinal cord. These findings suggested that activation of the ERK5 signaling pathway might contribute to morphine physical dependence and its specific pharmacological inhibitor BIX02188 could be a potential therapeutic choice for alleviation of morphine withdrawal symptoms in the future.

Substance P in the cerebrospinal fluid-contacting nucleus contributes to morphine physical dependence in rats

The cerebrospinal fluid-contacting nucleus (CSF-CN), distributes and localizes in the ventral periaqueductal central gray (PAG) of the brainstem, which may influence actual composition of the cerebrospinal fluid (CSF) for non-synaptic signal transmission via releasing or absorbing bioactive substances. Many experiments have demonstrated that substance P (SP), a substance that is shown to be up-regulated in CSF-CN, plays an important role in the development of inflammatory pain and neuropathic pain. Thus in the present study, we hypothesize that SP in CSF-CN might contribute to morphine dependence in rats, inhibiting SP with (D-Pro2, D-Phe7, D-Trp9)-SP intracerebroventricular (i.c.v.) injection reduce chronic morphine dependence and withdrawal. Rats were repeatedly injected with morphine in five escalating doses for morphine physical dependence. Morphine withdrawal-like behavioral signs and morphine analgesia behaviors were monitored after naloxone administration following i.c.v. injection of (D-Pro2, D-Phe7, D-Trp9)-SP. And SP-expression of CSF-CN was evaluated with dual-label immunofluorescent technique on morphine withdrawal in rats. After i.c.v. treatment with (D-Pro2, D-Phe7, D-Trp9)-SP, the naloxone-precipitated withdrawal symptoms were significantly attenuated, paw withdrawal threshold/thermal withdrawal latency (PWT/TWL) were increased, and SP-expression in CSF-CN was significantly reduced than control group. SP, known a neurotransmitter/neuromodulator of nociception, has also been implicated in the signs of opioid withdrawal. This study provides the first evidence that SP in CSF-CN contributes to morphine physical dependence and withdrawal, which may provide an important and specific role in mediating the motivational aspects of opiates withdrawal via CSF – the parenchyma of the brain, and may represent a novel pharmacological route such as SP inhibitor i.c.v. injection for the control of drug abuse.

β-Lactam antibiotic inhibits development of morphine physical dependence in rats

beta-Lactam antibiotics enhance cellular glutamate uptake. As increased glutamatergic transmission is a primary mediator of opiate dependence, we tested the hypothesis that a beta-lactam antibiotic (ceftriaxone) prevents development of morphine physical dependence in rats. Morphine (20 mg/kg) was injected twice daily for 10 days to induce physical dependence. Naloxone (10 mg/kg) administration 1, 48, and 96 h after the last morphine injection induced a withdrawal syndrome characterized by the appearance of wet-dog shakes, teeth chattering, eye blinking, jumping, and paw tremor. Ceftriaxone (150, 200 mg/kg) injected once daily during chronic morphine exposure inhibited each naloxone-precipitated withdrawal sign. Ceftriaxone efficacy persisted even after the 96 h-naloxone (10 mg/kg) injection. These results suggest that beta-lactam antibiotics inhibit processes leading to development of morphine physical dependence.

Effect of gene transfer of GLT-1, a glutamate transporter, into the locus coeruleus by recombinant adenoviruses on morphine physical dependence in rats.

There is a body of evidence implying the involvement of the central glutamatergic system in morphine dependence. We previously reported changes in the mRNA expression of a glial glutamate transporter GLT-1 in some brain regions of morphine-dependent and naloxone-precipitated withdrawal rats, and inhibition of morphine physical dependence by a glutamate transporter activator in mice. These findings support the possibility that glutamate transporters such as GLT-1 are involved in morphine dependence. In this study, we examined the effects of gene transfer of GLT-1 into the locus coeruleus (LC) by recombinant adenoviruses on morphine physical dependence in rats. We constructed recombinant adenoviruses that successfully delivered the GLT-1 gene in vitro and in vivo. Local overexpression of GLT-1 within the bilateral LC by the recombinant adenoviruses before implantation of the morphine pellet significantly inhibited various somatic signs of naloxone-precipitated morphine withdrawal, compared with the control. These results suggest that GLT-1 within the LC plays an inhibitory role in morphine physical dependence.

Ethanol-related adaptive changes and physical dependence in rats after exposure to ethanol

Adaptive changes that occur after chronic exposure to ethanol are an important component in the development of physical dependence. We have focused our research on ethanol-induced changes in the expression of several genes that may be important in adaptation. In this article, we describe adaptive changes at the level of the N-methyl- d-aspartate receptor, in the protein expression and activity of the Egr transcription factors, and in the expression of a novel gene of unknown function.

The dopamine D3/2 agonist 7-OH-DPAT attenuates the development of morphine tolerance but not physical dependence in rats.

The dopamine (DA) D3/2 agonist 7-OH-DPAT attenuates the acute antinociceptive, discriminative stimulus, locomotor activating, and reinforcing effects of mu agonists (for example, morphine). To examine the ability of 7-OH-DPAT to modulate the development of morphine tolerance and physical dependence in the rat. Morphine antinociception was assessed using a warm water tail-withdrawal procedure before and following chronic treatment with morphine (15 mg/kg)/7-OH-DPAT (0.3-3.0 mg/kg). Physical dependence was assessed following naloxone-precipitated (1.0 mg/kg) withdrawal in rats treated chronically with morphine (15 and 7.5 mg/kg)/7-OH-DPAT (1.0-10 mg/kg). 7-OH-DPAT attenuated the antinociceptive effects of morphine in both morphine naive and tolerant rats. Additionally, morphine tolerance was attenuated by the coadministration of 7-OH-DPAT in a dose- and time-dependent manner. The magnitude of the attenuation obtained when morphine and 7-OH-DPAT were administered at the same time was similar to that obtained when administration of these drugs was separated by 6 h, indicating that 7-OH-DPAT did not alter morphine pharmacokinetics. In rats rendered tolerant to morphine, the subsequent coadministration of morphine/7-OH-DPAT failed to reverse morphine tolerance, but did attenuate its further development. The level of physical dependence (number and frequency of withdrawal signs) was greater in rats treated with 15 than 7.5 mg/kg morphine. Under both treatment conditions, physical dependence was not altered by 7-OH-DPAT. In morphine-dependent (15 mg/kg) rats, 7-OH-DPAT (3.0 and 10 mg/kg) failed to precipitate withdrawal. The D3/2 agonist 7-OH-DPAT can attenuate the antinociceptive effects of morphine in both acute and chronic preparations as well as the development of morphine tolerance. 7-OH-DPAT does not, however, alter morphine physical dependence.

Precipitated and spontaneous withdrawal in rats tolerant to anandamide

There is evidence that cannabinoids cause tolerance and physical dependence in humans and animals. The aim of this work was to study whether the endogenous ligand for the cannabinoid receptor, arachidonylethanolamide (anandamide), induced behavioral tolerance and physical dependence in rats. Rats were injected with anandamide (20 mg/kg i.p.) daily for 2 weeks. To assess tolerance, on days 1, 8 and 15 of treatment rats were observed and behavior was tested. Two common methods were employed to assess physical dependence: interruption of anandamide dosing and vehicle substitution or administration of the cannabinoid CB1 receptor antagonist SR141716A (3 mg/kg i.p.). Full or partial tolerance developed to the classical behavioral effects elicited by the cannabinoids: hypothermia, catalepsy, hypomotility, decrease in stereotypic activity (rearing and grooming) and hindlimb splaying. No tolerance to anandamide was observed for reduced defecation. An abstinence syndrome appeared after abrupt cessation of cannabinoid intake and after withdrawal precipitated by SR141716A; the withdrawal signs were scratching, licking and biting, eating of feces, ptosis, arched back, wet dog shakes, head shakes, myoclonic spasms, writhing, forepaw fluttering, teeth chattering and piloerection. These findings indicate that the endogenous cannabinoid ligand, administered exogenously, induces both tolerance and physical dependence in rats.

Flunitrazepam and nordiazepam slowly released from silastic capsules induce physical dependence in rat

The rates of in vitro release of flunitrazepam (FN), nordiazepam (ND) and diazepam (DZ) from silastic capsules were compared and found to be in the following order: DZ > FN > ND. Rats that were implanted subcutaneously with capsules filled with FN or ND for 5 to 7 weeks before administering flumazenil (FLU) (40 mg/kg, i.v.) showed precipitated abstinence as measured by the Precipitated Abstinence Score (PAS) which included a rapid onset of clonic and tonic-clonic convulsions. Rats implanted with DZ also demonstrated significant PAS and seizures. Implantation of similar doses of DZ, FN and ND resulted in different plasma levels of parent benzodiazepines and their metabolites that corresponded with their in vitro release: DZ > FN > ND. These data indicate that, as for DZ, the capsule implantation is an effective method of producing physical dependence on FN and ND in the rat.

Production of antinociception by peripheral administration of [Lys7]dermorphin, a naturally occurring peptide with high affinity for mu-opioid receptors.

1. The opioid activity of the amphibian peptide, [Lys7]dermorphin, was studied in rats and mice. When administered intracerebroventricularly (i.c.v.), intravenously (i.v.) or subcutaneously (s.c.) it produced a long lasting analgesia. Its antinociceptive potency exceeded that of morphine 290 times by i.c.v. injection, and 25-30 times by peripheral administration. 2. The dose-response curves of [Lys7]dermorphin antinociception were shifted to the right by the pretreatment with naloxone (0.1 mg kg-1, s.c.) or with the mu 1-selective antagonist, naloxonazine (10 mg kg-1, i.v. 24 h before peptide injection). 3. The peptide also displayed potent antinociceptive effects in a chronic inflammatory pain model (rat Freund's adjuvant arthritis). In this pain model, systemic administration of the peptide raised the nociceptive threshold more in inflamed than in healthy paw. 4. High central and peripheral doses of [Lys7]dermorphin in rats produced catalepsy. The cataleptic response was antagonized by naloxone but left unchanged by naloxonazine pretreatment. 5. In rats and mice, central or peripheral administration of [Lys7]dermorphin induced a significantly slower development of tolerance to the antinociceptive effect than did morphine. 6. Upon naloxone precipitation of the withdrawal syndrome, [Lys7]dermorphin-dependent mice made fewer jumps and lost less weight than the morphine-dependent animals. Withdrawal hyperalgesia did not develop in [Lys7]dermorphin-dependent mice. 7. In conclusion, [Lys7]dermorphin seems to be a unique opioid peptide having a high penetration into the blood-brain barrier despite its low lipid solubility. This peptide causes fewer side-effects than other opioids and appears less likely than morphine to cause physical dependence in rats and mice.

P-474 - Studies on drug dependence (Rept. 226): Effects of serotonergic anxiolytics on the development of diazepam physical dependence in rats.

P-474 - Studies on drug dependence (Rept. 226): Effects of serotonergic anxiolytics on the development of diazepam physical dependence in rats.

Changes in striatal dopamine metabolism during the development of morphine physical dependence in rats: Observations using in vivo microdialysis

The present study examined the effect of prolonged morphine treatment on striatal dopamine (DA) release and metabolism, during the initial phase of the development of morphine dependence. Sprague-Dawley rats were implanted with chronic guides for microdialysis of the striatum. Morphine (two 75-mg pellets, subcutaneous implant) or placebo was given (12 hr) to pentobarbital anesthetized animals. Following recovery from anesthesia, morphine physical dependence was verified by the naloxone-evoked abstinence syndrome. Morphine produced significant increases in the dialysate level of DA and its metabolites (DOPAC and HVA) above baseline compared to placebo treatment. HVA levels began to increase immediately following morphine administration, whereas DA and DOPAC levels began to increase after a latency of one and three hr, respectively. Morphine effects on striatal DA metabolism included changes in the metabolic disposition of DA. Increases in HVA concentration accompanied increases in DOPAC concentration up to a threshold value of DOPAC efflux; further increases in DOPAC level were associated with decreases in HVA level. These in vivo data suggest that morphine-induced changes in the regulation of striatal dopaminergic function may be an important component of the development of physical dependence.

Daily dose of ethanol and the development and decay of acute and chronic tolerance and physical dependence in rats

Using behavioral and physiological measures, we compared the rates of development and decay of acute and chronic tolerance to ethanol (ET) and the severity of the withdrawal syndrome. Male rats were treated with 6, 9, or 12 g/kg/day ET or equicaloric dextrin maltose, delivered intragastrically. Although treatment duration varied, the total dose of ET was kept constant at 162 g/kg/ rat for the three groups. The effects of a cumulative test dose of ET or equicaloric dextrin maltose, after exposure to a total of 0, 42, 83, 126, and 162 g/kg ET, and at 3, 5, and 7 days after termination of the chronic treatments, were evaluated on rectal temperature, dowel performance, and tail-flick and startle responses. After the initial five tolerance tests, chronic treatments were discontinued and rats were tested in a modified open-field apparatus and for their startle response to an auditory stimulus at 8, 12, 16, 20, 32, and 40 h later. With all measures, little tolerance developed in the 6-g/kg/day group. On the other hand, development of chronic tolerance was fastest in rats treated with the 12-g/kg/dose of ET. Chronic tolerance did not develop to ET's depressant effect on the startle response. Acute tolerance declined with chronicity of treatment in animals given the largest daily dose of ET. During withdrawal, and in contrast to the dextrin maltose-treated animals, there was impairment in all measures taken during the modified open-field test and hypersensitivity of the startle response for all three ET-treated animals. Greatest behavioral impairment occurred in animals treated with 12 g/kg/ day, and some impairment was still evident 40 h after the last dose of ET. Thus, the severity of the withdrawal syndrome was greatest in the group displaying the most acute and chronic tolerance.